Materials and methods involving conditional retention domains

ABSTRACT

Materials and methods involving conditional retention domains (CRDs) are disclosed. Also disclosed are fusion proteins containing CRDs and cells expressing such fusion proteins. In addition, the invention provides novel methods for producing target proteins in vivo using fusion proteins containing conditional retention domains and methods for identifying novel CRDs.

BACKGROUND OF THE INVENTION

[0001] A number of important applications, including for example, genetherapy, production of biological materials and materials and methodsfor biological research, depend on the ability to induce cells toproduce proteins of therapeutic, commercial, or experimental value. Avariety of regulatable expression systems have been developed, includingsystems involving allostery-based switches triggered by tetracycline,RU486 or ecdysone, as well as dimerization-based switches triggered bydimerizing agents such as rapamycin, coumermycin, dimers of FK506,synthetic FKBP-binders and/or CsA, or analogs thereof. See e.g.Clackson, “Controlling mammalian gene expression with small molecules”Current Opinion in Chemical Biology, 1:210-218, 1997. In theseexpression systems, protein production is regulated at thetranscriptional level. An inherent limitation of all such systems is theinability to achieve fine temporal control over secretion of the targetprotein. For example, secretion of maximal, therapeutic levels of theprotein is delayed by many hours or even days until the transcribed mRNAaccumulates to levels high enough to produce significant amounts ofsecreted protein. Likewise, secretion cannot return to low baselinelevels following removal of the inducing drug until the mRNA iscompletely degraded, which may also take many hours or days. For manyapplications this level of control is not sufficient; in theseinstances, it would be desirable to induce protein production on a muchmore rapid time scale than that achievable using transcription-basedmethods.

SUMMARY OF THE INVENTION

[0002] This invention takes a unique approach to the regulatedproduction of a target protein, based not on regulated transcription,but on regulated release or secretion of the target protein.Compositions and methods of this invention are useful in biologicalresearch and in gene therapy applications.

[0003] Key features of the invention include conditional retentiondomains (“CRDs”), fusion proteins containing them, ligands which bind tothe CRDs and permit release or secretion of the fusion proteins,recombinant nucleic acids encoding such fusion proteins, vectorscontaining such recombinant nucleic acids, cells transduced with thesevectors and other materials and important methods involving such. Keyfusion proteins of the invention contain at least two mutuallyheterologous domains, one of which being a CRD.

[0004] More specifically, the fusion proteins of this invention aredesigned to contain at least one conditional retention domain (CRD) andat least one additional domain that is heterologous thereto, usuallywith a secretory signal sequence. Proteins containing a secretory signalsequence are translated in the endoplasmic reticulum (ER) and then passthrough other secretory compartments such as the cis, medial and transGolgi on their way to being secreted. However, proteins containing oneor more CRDs are, as a rule, retained in the secretory machinery exceptin the presence of a ligand which binds to the protein. Illustrativeexamples of CRDs include retinol binding proteins and human FKBP 12mutants such as F36M hFKBP12, as are discussed in detail below.Concatenation of multiple CRDs may allow the user to modulate the degreeof aggregation or retention.

[0005] Typically the fusion protein also contains a secretory signalsequence to target the fusion protein to a secretory compartment such asthe ER or any part of the Golgi apparatus. Many secretory signalsequences are known. Human growth hormone, for example, is the source ofa secretory signal sequence suitable for use in this invention.

[0006] Additionally, it is preferred in many embodiments that the fusionprotein further contain an enzymatic cleavage site such that a portionof the fusion protein containing the CRD can be cleaved from a portionof the fusion protein containing a peptide sequence heterologous to theCRD. Preferably the enzymatic cleavage site comprises a peptide sequencerecognized by a trans-Golgi specific endoprotease such as furin. Forinstance, a cleavage site for furin is provided by the peptide sequenceSARNRQKR (SEQ ID NO. 1).

[0007] The portion of the fusion protein which is heterologous to theCRD may comprise any protein or protein domain of interest to thepractitioner. For instance, the heterologous portion may comprise atarget protein such as insulin, parathyroid hormone or beta-endorphin.

[0008] To illustrate this further, one typical fusion protein of theinvention comprises a signal sequence, a conditional retention domain, afurin cleavage site, and a polypeptide sequence comprising a selectedtarget protein sequence. An example of such a fusion protein comprises,in N-terminal to C-terminal order, a signal sequence from human growthhormone, three F36M hFKBP 12 domains, a human stromelysin-3 furincleavage site, and a selected target protein sequence. Fusion proteinsmay also contain several target proteins each separated by an enzymaticcleavage site. For example, such a fusion protein might contain a signalsequence from human growth hormone, one or more copies of a CRD such asF36M hFKBP 1{overscore (2)}, a furin cleavage site, a target protein,another furin cleavage site and another target protein. This type ofconstruct allows for simultaneous release of more than one targetprotein.

[0009] In addition, the fusion proteins of this invention may optionallycomprise a lysosomal targeting signal or other polypeptide sequencetargeting it for degradation. By locating such a peptide sequencetogether with the CRD(s) on one side of the cleavage site and theselected target polypeptide on the other side of the cleavage site, onecan help assure cellular removal of the CRD-containing portion of thefusion protein.

[0010] One object of the invention is thus the fusion proteins describedherein.

[0011] Another object of the invention is the recombinant nucleic acidsencoding such fusion proteins. Those recombinant nucleic acids may beoperably linked to an expression control sequence permitting theirexpression in host cells into which they have been transduced, or whichotherwise contain them. Any promoter may be used to drive expression ofthese fusion proteins, including strong promoters like the CMV enhancer,other viral promoters such as the RSV promoter or tissue specificpromoters like the MCK enhancer.

[0012] Another object is a vector containing a recombinant nucleic acidof the invention, generally operably linked to an expression controlsequence. Such vectors include “viral” vectors which contain part or allof a viral genome in addition to the recombinant nucleic acid encodingthe fusion protein of this invention. Viral vectors can be designed andused for the production of recombinant viruses harboring a recombinantnucleic acid of this invention. A wide variety of such viral systems areknown in the art and may be adapted to the practice of this invention,including e.g. adenovirus, AAV, retrovirus, hybrid adeno-AAV, lentivirusand others.

[0013] Recombinant nucleic acids of this invention may be transducedinto host cells by any available means e.g. in order to render thosecells capable of regulated secretion of a target protein. The cells arepreferably eukaryotic cells, generally are animal cells, and in manyembodiments are mammalian, whether human or non-human. The cells may betransduced in situ within their host organism, or they may be transducedwhile being maintained in vitro. The cells may be primary cells or maybe from a cell line.

[0014] The invention thus provides methods for rendering a cell capableof regulated secretion of a target protein which involves introducinginto the cell a recombinant nucleic acid of this invention to yieldengineered cells which can express the encoded fusion protein. Therecombinant nucleic acid may be introduced in viral or other form intocells maintained in vitro or into cells present within an organism. Theresultant engineered cells and their progeny containing one or more ofthese recombinant nucleic acids may be used in a variety of importantapplications discussed elsewhere, including human gene therapy,analogous veterinary applications, the creation of cellular or animalmodels (including transgenic applications), assay applications, and theproduction of a desired protein in vitro, e.g. for recovery and use.Such cells are useful, for example, in methods involving the addition ofa ligand, preferably a cell permeant ligand, to the cells (oradministration of the ligand to an organism containing the cells) toregulate secretion of a target protein. Particularly important animalmodels include rodent (especially mouse and rat) and non-human primatemodels. In human gene therapy applications, the cells will generally behuman and the peptide sequence of each of the various domains present inthe fusion proteins will preferably be, or be derived from, a peptidesequence of human origin, to the extent possible.

[0015] The invention also provides methods for identifying novel CRDs.CRDs may be identified by two hybrid type methods, in which agenetically engineered host cell is provided which comprises (a) areporter gene linked to a regulatable expression control element, and(b) a recombinant nucleic acid comprising a polylinker linked to tworecombinant nucleic acid sequences, the first recombinant nucleic acidsequence encoding a DNA binding domain and the second recombinantnucleic acid sequence encoding a transcription activation domain,wherein association of the DNA binding domain with the transcriptionactivation domain activates expression of the reporter gene. Asdescribed herein, the construct contains a single polylinker linked totwo independent translational cassettes. This allows for expression oftwo fusion proteins, one with a DNA binding domain and the other with atranscription activation domain, each linked to an identical CRDcandidate. In addition, genetically engineered host cells are providedwhich comprise (a) a reporter gene linked to a regulatable expressioncontrol element, (b) a first recombinant nucleic acid encoding a fusionprotein comprising a transcription activation domain linked to acandidate conditional retention domain, (c) a second recombinant nucleicacid encoding a fusion protein containing a DNA binding domain linked tothe candidate conditional retention domain wherein association of thefusion proteins activates expression of the reporter gene.

[0016] The invention further provides methods for identifying a ligandcapable of binding to a conditional retention domain. See, “Methods foridentifying CRDs”, part 3, page 46 et seq, below. One such method usescells genetically engineered to express a reporter gene whenCRD-containing aggregates are disaggregated by an appropriate ligand.The method involves the following steps: (a) contact the geneticallyengineered cells with candidate ligands under suitable conditionspermitting gene expression, (b) observe the presence and/or amount ofexpression of the reporter gene, and (c) correlate the presence and/oramount of reporter gene expression with contact of cells with one ormore candidate ligands.

[0017] The invention also provides methods for screening directly forCRDs which enable ligand-dependent secretion of a target protein orligand-dependent localization of a membrane protein. For these screeningassays, fusion proteins are expressed which encode members of a libraryof candidate CRDs linked to a signal sequence and an enzymatic cleavagesite. These domains are further linked to either a secreted targetprotein or the extracellular and membrane domain of a membrane protein.The fusion proteins are expressed under conditions permitting secretionof the target protein or localization of the membrane protein. Cellscontaining the fusion proteins are treated with a ligand that binds theCRD, and then the ligand-dependent presence of the secreted protein ormembrane protein is assessed. Secretion of the target protein and/orlocalization of the membrane protein is then correlated with one or moreindividual members of the CRD library.

BRIEF DESCRIPTION OF THE FIGURES

[0018]FIG. 1: General design of fusion proteins for use in thisinvention, containing, from amino- to carboxy-terminus, a secretionsignal sequence, a “conditional retention domain”, a protease cleavagesite, and the secreted target protein of interest.

[0019]FIG. 2: Constructs used to make CRD-containing fusion proteins.FIG. 2A: F36M-EGFP fusion proteins; FIG. 2B: F36M-hGH fusion proteins;FIG. 2C: EGFP-F36M-hGH fusion proteins; FIG. 2D: F36M-insulin fusionproteins; FIG. 2E: LNGFR-F36M fusion proteins.

[0020]FIG. 3: Ligand dependent secretion of hGH. Levels of hGH secretedinto the culture medium of transiently transfected (FIG. 3A) or stablytransfected (FIG. 3B) HT1080 cells in the absence and presence ofligand.

[0021]FIG. 4: Immunoblots of cell lysates and supernatants prepared fromthe HT88 cells incubated in the presence or absence of ligand for 2hours. The samples were immunoblotted with anti-hGH and anti-FKBPantibodies.

[0022]FIG. 5: Dose-dependence of hGH secretion from HT88 cells inresponse to ligand (FIG. 5A). Time course of accumulation of secretedhGH in the culture medium (FIG. 5B).

[0023]FIG. 6: Kinetics of secretion in response to ligand. FIG. 6A GroupA: the constitutive rate of secretion from the cells. Group B: secretionfrom cells not previously exposed to ligand. Group C: cells exposed toligand following a large bolus release of hGH. FIG. 6B shows the amountof hGH released by incubation with maximal concentration of ligand. FIG.6c shows the amount of hGH secreted following addition of sub-maximalconcentrations of ligand.

[0024]FIG. 7: Effect of varying the number of CRDs on hGH secretion. hGHsecretion was measured following addition of ligand in cell linesexpressing fusion proteins containing varying numbers of CRDs.

[0025]FIG. 8: Regulated secretion of insulin. Levels of insulinsecretion were measured in transiently transfected HT1080 cells treatedwith varying concentrations of AP21998.

[0026]FIG. 9: Regulated expression of a membrane tethered protein. 3, 4,or 6 copies of F(36M) were fused to the extracellular and transmembraneportions of the low-affinity nerve growth factor receptor (LNGFR; FIG.3E). Surface expression was assessed by FACS analysis using anti-LNGFRantibodies.

[0027]FIG. 10: Constructs useful for screening for novel CRDs. A.Candidate DNA sequences may be cloned into the polylinker foridentifying CRDs that induce ligand-dependent secretion of hGH. B.Candidate DNA sequences may be cloned into the polylinker foridentifying CRDs that induced ligand-dependent localization of p75. C.Construct used for “two hybrid” style assay, in which fusion proteinscontaining CRDs cause association of the DNA binding domain andtranscription activation domain to induce transcription.

[0028]FIG. 11: Ligand-mediated regulation of insulin and glucose levelsin vivo. (A) Insulin and glucose levels were measured in mice implantedwith FKBP(F36M)-insulin-containing constructs before and afteradministration of AP22542. (B) Levels of serum glucose were measured inmice implanted with FKBP(F36M)-insulin-containing constructs at varioustime points following administration of AP22542.

DETAILED DESCRIPTION

[0029] Definitions:

[0030] For convenience, the intended meaning of certain terms andphrases used herein are provided below.

[0031] “Capable of selectively hybridizing” means that two DNA moleculesare susceptible to detectable hybridization with one another, despitethe presence of other DNA molecules, under hybridization conditionswhich can be chosen or readily determined empirically by thepractitioner of ordinary skill in this art. Such treatments includeconditions of high stringency such as washing extensively with bufferscontaining 0.2 to 6×SSC, and/or containing 0.1% to 1% SDS, attemperatures ranging from room temperature to 65-75° C. See for exampleF. M. Ausubel et al., Eds, Short Protocols in Molecular Biology, Units6.3 and 6.4 (John Wiley and Sons, New York, 3d Edition, 1995).

[0032] “Cells”, “host cells” or “recombinant host cells” refer not onlyto the particular cells under discussion, but also to their progeny.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, such progeny may not, infact, be identical to the parent cell, but are still included within thescope of the term as used herein.

[0033] “Cell line” refers to a population of cells capable of continuousor prolonged growth and division in vitro. Often, cell lines are clonalpopulations derived from a single progenitor cell. It is further knownin the art that spontaneous or induced changes can occur in karyotypeduring storage or transfer of such clonal populations. Therefore, cellsderived from a given cell line may not be precisely identical to theancestral cells or cultures, and the cell line referred to includes suchvariants.

[0034] “Composite”, “fusion”, and “recombinant” denote a material suchas a nucleic acid, nucleic acid sequence or polypeptide which containsat least two constituent portions which are mutually heterologous in thesense that they are not otherwise found directly (covalently) linked innature, i.e., are not found in the same continuous polypeptide or genein nature, at least not in the same order or orientation or with thesame spacing present in the composite, fusion or recombinant product.Typically, such materials contain components derived from at least twodifferent proteins or genes or from at least two non-adjacent portionsof the same protein or gene. In general, “composite” refers to portionsof different proteins or nucleic acids which are joined together to forma single functional unit, while “fusion” generally refers to two or morefunctional units which are linked together. “Recombinant” is generallyused in the context of nucleic acids or nucleic acid sequences.

[0035] A “coding sequence” or a sequence which “encodes” a particularpolypeptide or RNA, is a nucleic acid sequence which is transcribed (inthe case of DNA) and translated (in the case of mRNA) into a polypeptidein vitro or in vivo when placed under the control of an appropriateexpression control sequence. The boundaries of the coding sequence aregenerally determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxy) terminus. A coding sequencecan include, but is not limited to, cDNA from procaryotic or eukaryoticmRNA, genomic DNA sequences from procaryotic or eukaryotic DNA, andsynthetic DNA sequences. A transcription termination sequence willusually be located 3′ to the coding sequence.

[0036] A “construct”, e.g., a “nucleic acid construct” or “DNAconstruct”, refers to a nucleic acid or nucleic acid sequence.

[0037] “Derived from” denotes a peptide or nucleotide sequence selectedfrom within a given sequence. A peptide or nucleotide sequence derivedfrom a named sequence may further contain a small number ofmodifications relative to the parent sequence, in most casesrepresenting deletion, replacement or insertion of less than about 15%,preferably less than about 10%, and in many cases less than about 5%, ofamino acid residues or bases present in the parent sequence. In the caseof DNAs, one DNA molecule is also considered to be derived from anotherif the two are capable of selectively hybridizing to one another.Polypeptides or polypeptide sequences are also considered to be derivedfrom a reference polypeptide or polypeptide sequence if any DNAsencoding the two polypeptides or sequences are capable of selectivelyhybridizing to one another. Typically, a derived peptide sequence willdiffer from a parent sequence by the replacement of up to 5 amino acids,in many cases up to 3 amino acids, and very often by 0 or 1 amino acids.A derived nucleic acid sequence will differ from a parent sequence bythe replacement of up to 15 bases, in many cases up to 9 bases, and veryoften by 0-3 bases. In some cases the amino acid(s) or base(s) is/areadded or deleted rather than replaced.

[0038] “Domain” refers to a portion of a protein or polypeptide. In theart, the term “domain” may refer to a portion of a protein having adiscrete secondary structure. However, as will be apparent from thecontext used herein, the term “domain” as used in this document does notnecessarily connote a given secondary structure. Rather, a peptidesequence is referred to herein as a “domain” simply to denote apolypeptide sequence from a defined source, or having or conferring anintended or observed activity. Domains can be derived from naturallyoccurring proteins or may comprise non-naturally-occurring sequence.

[0039] “Expression control element”, or simply “control element”, refersto DNA sequences, such as initiation signals, enhancers, promoters andsilencers, which induce or control transcription of DNA sequences withwhich they are operably linked. Control elements of a gene may belocated in introns, exons, coding regions, and 3′ flanking sequences.Some control elements are “tissue specific”, i.e., affect expression ofthe selected DNA sequence preferentially in specific cells (e.g., cellsof a specific tissue), while others are active in many or most celltypes. Gene expression occurs preferentially in a specific cell ifexpression in this cell type is observably higher than expression inother cell types. Control elements include so-called “leaky” promoters,which regulate expression of a selected DNA primarily in one tissue, butcause expression in other tissues as well. Furthermore, a controlelement can act constitutively or inducibly. An inducible promoter, forexample, is demonstrably more active in response to a stimulus than inthe absence of that stimulus. A stimulus can comprise a hormone,cytokine, heavy metal, phorbol ester, cyclic AMP (cAMP), retinoic acidor derivative thereof, etc. A nucleotide sequence containing one or moreexpression control elements may be referred to as an “expression controlsequence”.

[0040] “Gene” refers to a nucleic acid molecule or sequence comprisingan open reading frame and including at least one exon and (optionally)one or more intron sequences.

[0041] “Genetically engineered cells” denotes cells which have beenmodified by the introduction of recombinant or heterologous nucleicacids (e.g. one or more DNA constructs or their RNA counterparts) andfurther includes the progeny of such cells which retain part or all ofsuch genetic modification.

[0042] “Heterologous”, as it relates to nucleic acid or peptidesequences, denotes sequences that are not normally joined together,and/or are not normally associated with a particular cell. Thus, a“heterologous” region of a nucleic acid construct is a segment ofnucleic acid within or attached to another nucleic acid molecule that isnot found in association with the other molecule in nature. For example,a heterologous region of a construct could include a coding sequenceflanked by sequences not found in association with the coding sequencein nature. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,synthetic sequences having codons different from the native gene).Similarly, in the case of a cell transduced with a nucleic acidconstruct which is not normally present in the cell, the cell and theconstruct would be considered mutually heterologous for purposes of thisinvention.

[0043] “Interact” refers to directly or indirectly detectableinteractions between molecules, such as can be detected using, forexample, a yeast two hybrid assay or by immunoprecipitation. The term“interact” encompasses “binding” interactions between molecules.Interactions may be, for example, protein-protein, protein-nucleic acid,protein-small molecule or small molecule-nucleic acid in nature.

[0044] “Nucleic acid” refers to polynucleotides such as deoxyribonucleicacid (DNA), and, where appropriate, ribonucleic acid (RNA). The termshould also be understood to include derivatives, variants and analogsof either RNA or DNA made from nucleotide analogs, and, as applicable tothe embodiment being described, single (sense or antisense) anddouble-stranded polynucleotides.

[0045] A “polylinker”, also sometimes referred to as a “multiple cloningsite” is a region within a vector which contains multiple sites forrestriction enzyme cleavage, thus rendering the vector suitable forcloning of exogenous genes.

[0046] “Protein”, “polypeptide” and “peptide” are used interchangeably.

[0047] A “recombinant virus” is a virus particle in which the packagednucleic acid contains a heterologous portion.

[0048] The “secretory machinery” (also called secretory apparatus) ofthe cell refers to the cellular compartments to which secreted andmembrane proteins are targeted and processed. These compartments includethe endoplasmic reticulum (ER) and the cis, medial and trans Golgi. Inthis document, the term ER is often used generically to mean “secretorycompartment.”

[0049] A “target protein” is a protein of interest, the secretion ofwhich is modulated according to the methods of the invention. The targetprotein can be, for example, a hormone, an endorphin, etc.

[0050] “Transfection” means the introduction of a naked nucleic acidmolecule into a recipient cell. “Infection” refers to the processwherein a nucleic acid is introduced into a cell by a virus containingthat nucleic acid. A “productive infection” refers to the processwherein a virus enters the cell, is replicated, and is then releasedfrom the cell (sometimes referred to as a “lytic” infection).“Transduction” encompasses the introduction of nucleic acid into cellsby any means.

[0051] “Transgene” refers to a nucleic acid sequence which has beenintroduced into a cell. Daughter cells deriving from a cell in which atransgene has been introduced are also said to contain the transgene(unless it has been deleted). The polypeptide or RNA encoded by atransgene may be partly or entirely heterologous, i.e., foreign, withrespect to the animal or cell into which it is introduced.Alternatively, the transgene can be homologous to an endogenous gene ofthe transgenic animal or cell into which it is introduced, but isdesigned to be inserted, or is inserted, into the animal's genome insuch a way as to alter the genome of the cell into which it is inserted(e.g., it is inserted at a location which differs from that of thenatural gene). A transgene can also be present in an episome. Atransgene can include one or more expression control elements and anyother nucleic acid, (e.g. intron), that may be necessary or desirablefor optimal expression of a selected coding sequence.

[0052] The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is an episome, i.e., a nucleic acid capable ofextra-chromosomal replication. Often vectors are used which are capableof autonomous replication and/or expression of nucleic acids to whichthey are linked. Vectors capable of directing the expression of anincluded gene operatively linked to an expression control sequence canbe referred to as “expression vectors”. Expression vectors are typicallyin the form of “plasmids” which refer generally to circular doublestranded DNA loops which, in their vector form are not bound to thechromosome. In the present specification, “plasmid” and “vector” areused interchangeably as the plasmid is the most commonly used form ofvector. However, the invention is intended to include such other formsof vectors which serve equivalent functions and which are or becomeknown in the art. Viral vectors are nucleic acid molecules containingviral sequences which can be packaged into viral particles.

[0053] Conditional Retention Domains:

[0054] A conditional retention domain is any domain which is retained inthe ER or other secretory compartment in the absence of ligand and isreleased from the secretory machinery when ligand is bound, i.e. in thepresence of ligand. The use of CRDs is considered to take advantage ofthe phenomenon of ER “quality control”, whereby proteins that areincorrectly folded or aggregated are retained in the ER rather thantraveling to the Golgi. Eventually, most misfolded proteins aredegraded, but others have been observed to accumulate in substantialsteady-state amounts (eg. the VSV-G protein: A. M. de Silva et al.(1990) J. Cell Biol. 111, 857-866. See also, Kopito, R. R. (1997) Cell88, 427-430). Several types of domains can function as conditionalretention domains:

[0055] 1) The CRD can be a Natural Example of a Protein that is Retainedin the Secretory Machinery in the Absence of a Particular SmallMolecule.

[0056] An example of this type of conditional retention domain is or isderived from retinol binding protein (RBP). Retinol binding protein is aserum protein of approximately 20 kD that is a specific carrier forretinol (Vitamin A) (Melhus, H. et al. (1992) J Biol. Chem. 267,12036-12041). It is retained in the ER in complex with another protein,transerythrin. Upon binding of retinol to RBP, the complex is releasedfrom its molecular chaperone and is free to enter the Golgi apparatus.Thus, the retinol binding protein acts as a CRD which is retained in theER in the absence of ligand and secreted in its presence. Althoughretinol binding protein is expressed primarily in hepatocytes, it isgenerally useful as a CRD, since several groups have shown thatretinol-mediated secretion of RBP is cell-type independent and requiresno hepatocyte specific cofactors (see, e.g. Melhus et al., J. Biol.Chem. 267:12036-12041, 1992.)

[0057] Another example of a protein that is retained in the ER in theabsence of a small molecule ligand is IgM. Retention of soluble μ chainsin the ER is dependent on a single unpaired cysteine residue. Althoughsecretion of IgM normally requires binding of light chains to the μheavy chain, secretion of IgM intermediates can be induced by additionof 2-mercaptoethanol or other reducing agents (Alberini et al., Nature347:485-487, 1990). Thus, soluble μ chains can function as CRDs whichare secreted in the presence of a thiol-reactive small molecule.

[0058] 2) The CRD can be an Engineered Mutant of a Natural Protein,Chosen because it has the Property of being Selectively Retained in theAbsence of a Given Small Molecule.

[0059] It is known that mutations that destabilize proteins can lead toER retention. Without wishing to be bound to any one theory, includingthat theory, we have observed that some mutations at human FKBP Phe36lead to proteins that are poorly expressed (eg. F36A), probably due toinstability. Such proteins are thought to be retained to some extent inthe secretory apparatus. Using a high affinity ligand that binds to theprotein to permit ER exit.

[0060] 3) The CRD can be a Protein that Self-aggregates in a SmallMolecule-reversible Manner.

[0061] It is known that large protein aggregates are retained in the ER.In such cases, ER retention occurs because of formation of aggregatesrather than due to misfolding of proteins. A naturally occurring exampleof aggregation-dependent ER retention is found in the Z mutation ofα₁-antitrypsin. In the secreted M form of this plasma protease, aglutamic acid residue is located at position 342 in the reactive centerloop of the molecule. In the mutant Z form, this glutamic acid issubstituted by lysine; this substitution allows the reactive loop toinsert itself into the A-sheet of an adjacent α₁-antitrypsin molecule,forming linear, transport-incompetent aggregates. The aggregatesaccumulate in the ER, but can be released by addition of a peptide whichinserts into the A-sheet and prevents polymerization (Hammond andHelenius, Current Opinion in Cell Biology 7:523-529, 1995; Lomas et al.,Nature 357:605-607, Jun. 18, 1992).

[0062] The mutant form of α-galactosidase A that is found in Fabrylymphoblasts provides an additional example of small-molecule dependentrelease of aggregates from the ER. Whereas the wild-type form of theenzyme is efficiently routed through the secretory pathway, the mutantprotein aggregates in the endoplasmic reticulum, contributing, at leastin part, to enzyme deficiency in Fabry patients. Recently, Fan et alreported that addition of 1-deoxy-galactonojirimycin (DGJ), acompetitive inhibitor of α-galactosidase A, enhances α-galactosidase Aactivity in Fabry lymphoblasts by acting as a “chemical chaperone”, thusaccelerating transport and processing of the mutant enzyme (Fan et al.,Nature Medicine 5:112-115, 1999).

[0063] In a preferred embodiment, the CRD is derived from human FKBP12.In particular, the FKBP mutant F36M functions as a conditional retentiondomain when fused to a signal sequence and heterologous target sequencein mammalian cells. In the absence of ligand, fusion proteins containingFKBP F36M and a signal sequence self-aggregate and accumulate in theendoplasmic reticulum. Upon addition of ligand, the fusion proteindisaggregates and transits through the ER, resulting in secretion of thefusion protein or cleavage products thereof. Another FKBP mutant whichfunctions as a CRD is FKBP W59V.

[0064] Ligands for CRDs:

[0065] A wide variety of ligands, including both naturally occurring andsynthetic substances, can be used in this invention to effectdisaggregation and/or secretion of the fusion protein molecules from thesecretory machinery. Criteria for selecting a ligand are: (A)physiologic acceptability of the ligand (i.e., the ligand lacks unduetoxicity towards the cell or animal for which it is to be used), (B)reasonable therapeutic dosage range, (C) suitability for oraladministration (i.e., suitable stability in the gastrointestinal systemand absorption into the vascular system), for applications in wholeanimals, including gene therapy applications, (D) ability to crosscellular and other membranes, as necessary, (E) reasonable bindingaffinity for the CRD (for the desired application), and (F) efficacy instimulating transit of the fusion protein. Preferably the compound isrelatively physiologically inert, but for its affinity for the CRD. Theless the ligand binds to native proteins or other materials within thecells to be targeted, the better the response will normally be.Preferably the ligand will be other than a peptide or nucleic acid, andwill preferably have a molecular weight of less than about 5000 Daltons,more preferably less than about 1200 Daltons.

[0066] In various embodiments where a ligand binding domain for acandidate ligand is endogenous to the cells to be engineered, it isoften desirable to alter the peptide sequence of the ligand bindingdomain and to use a ligand which discriminates between the endogenousand engineered ligand binding domains. Such a ligand should bindpreferentially to the engineered ligand binding domain relative to anaturally occurring peptide sequence, e.g., from which the modifieddomain was derived. This approach can avoid untoward intrinsicactivities of the ligand. Significant guidance and illustrative examplestoward that end are provided in the various references cited herein.

[0067] Substantial structural modification of a ligand for a ligandbinding domain is permitted, so long as the modified compound stillfunctions as a ligand for the ligand binding domain of interest, i.e.,so long as the compound possesses sufficient binding affinity andspecificity to function as disclosed herein. Some of the compounds willbe macrocyclics, e.g. macrolides, although linear and branched compoundsmay be preferred in specific embodiments. Suitable binding affinitieswill be reflected in Kd values well below 10⁻⁴, preferably below 10⁻⁶,more preferably below about 10⁻⁷, although binding affinities below 10⁻⁹or 10⁻¹⁰ are possible, and in some cases will be most desirable.

[0068] Illustrative examples of ligand binding domain/ligand pairsinclude retinol binding protein or variants thereof and retinol orderivatives thereof; cyclophilin or variants thereof and cyclosporin oranalogs thereof; FKBP or variants thereof and FK506, FK520, rapamycin,analogs thereof or synthetic FKBP ligands. In the case of a ligandbinding domain comprising or derived from an immunophilin orcyclophilin, the complex of the ligand with the ligand binding domainwill desirably not bind specifically to calcineurin or FRAP. A widevariety of FK506 derivatives and synthetic FKBP ligands are known whichdo not have observable immunosuppressive activity. Likewise, a varietyof rapamycin analogs are known which bind to FKBP but are notimmunosuppressive. See e.g. WO 98/02441 for non-immunosuppressiverapalogs. Those and other ligands can be used as well, depending on thechoice of CRD. Numerous assays are known in the art for identifyingligands which bind to CRDs that are identified through screening, asdescribed below.

[0069] Ligand binding domain/ligand pairs are illustrated by FKBPdomains, e.g. F36M FKBP, and FKBP ligands. In general, it is preferredthat the ligand bind preferentially to a mutated (i.e., having a peptidesequence not naturally occurring in the cells to be engineered) FKBPrelative to wild-type FKBP. Ligands for FKBP proteins, including F36MFKBP, can comprise or be derived from a naturally occurring FKBP ligandsuch as rapamycin, FK506 or FK520, or a synthetic FKBP ligand, e.g. asdisclosed in PCT/US95/10559; Holt, et al., J. Amer. Chem. Soc., 1993,115, 9925-9938; Holt, et al., Biomed. Chem. Lett., 1993, 4, 315-320;Luengo, et al., Biomed. Chem. Lett., 1993, 4, 321-324; Yamashita, etal., Biomed. Chem. Lett., 1993, 4, 325-328; PCT/US94/08008. See also EP0 455 427 A1; EP 0 465 426 A1; US 5,023,26; WO 92/00278; WO 94/18317; WO97/31898; WO 96/41865; and Van Duyne et al (1991) Science 252, 839.

[0070] Illustrative types of ligands for FKBP-derived ligand bindingdomains include the following Genus I:

[0071] where

[0072] n=1 or 2;

[0073] X=O, S, NH or CH₂;

[0074] B¹ and B² are independently H or aliphatic, heteroaliphatic, arylor heteroaryl as those terms are defined below, usually containing oneto about 12 carbon atoms (not counting carbon atoms of optionalsubstituents);

[0075] Y=O, S, NH, —NH(C═O)—, —NH(C═O)—O—, —NH(SO₂)— or NR³, orrepresents a direct, i.e. covalent, bond from R² to carbon 9;

[0076] R¹, R², and R³ are aliphatic, heteroaliphatic, aryl orheteroaryl, usually containing one to about 36 carbon atoms (notcounting carbon atoms of optional substituents);

[0077] two or more of B¹, B² and R² may be covalently linked to form aC3-C7 cyclic or heterocyclic moiety; and,

[0078] The term “aliphatic” as used herein includes both saturated andunsaturated straight chain, branched, cyclic, or polycyclic aliphatichydrocarbons, which are optionally substituted with one or moresubstituents.

[0079] The term “substituents” includes aliphatic, aryl, heteroaryl andheterocyclic moietites, which may themselves be substituted, as well asfunctional groups such as R⁸, —OR⁸, —SR⁸, —CN,—CHO, ═O, —COOH, —COR⁸,OS(O)₂ R⁸, —SO₂—NHR⁸, —NHSO₂ R⁸, sulfate, sulfonate, (or ester,carbamate, urea, oxime or carbonate thereof), —NH₂ (or substitutedamine, amide, urea, carbamate or guanidino derivative therof), halo,trihaloalkyl, —SO₂—CF₃, and —OSO₂F, where R⁸ may be H, aliphatic, aryl,heteroaryl or heteroaliphatic. Aliphatic, heteraliphatic, aryl andheterocyclic substituents may themselves be substituted or unsubstituted(e.g. mono-, di- and tri-alkoxyphenyl; methylenedioxyphenyl orethylenedioxyphenyl; halophenyl; or -phenyl-C(Me)₂—CH₂—O—CO—[C3-C6]alkyl or alkylamino). Additional examples of substituents areillustrated by the specific embodiments shown in the Examples whichfollow. (Unless otherwise specified, the alkyl, other aliphatic, alkoxyand acyl groups preferably contain 1-8, and in many cases 1-6,contiguous aliphatic carbon atoms).

[0080] The term “aliphatic” is thus intended to include alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties.

[0081] As used herein, the term “alkyl” includes both straight andbranched alkyl groups. An analogous convention applies to other genericterms such as “alkenyl”, “alkynyl” and the like. Furthermore, as usedherein, the language “alkyl”, “alkenyl”, “alkynyl” and the likeencompasses both substituted and unsubstituted groups.

[0082] The term “alkyl” refers to groups usually having one to eight,preferably one to six carbon atoms. For example, “alkyl” may refer tomethyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, pentyl, isopentyl tert-pentyl, hexyl, isohexyl, and thelike. Suitable substituted alkyls include, but are not limited to,fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl,3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, and thelike.

[0083] The term “alkenyl” refers to groups usually having two to eight,preferably two to six carbon atoms. For example, “alkenyl” may refer toprop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl,hex-5-enyl, 2,3-dimethylbut-2-enyl, and the like. The language“alkynyl,” which also refers to groups having two to eight, preferablytwo to six carbons, includes, but is not limited to, prop-2-ynyl,but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl,hex-5-ynyl, and the like.

[0084] The term “cycloalkyl” as used herein refers to groups havingthree to seven, preferably three to six carbon atoms. Suitablecycloalkyls include, but are not limited to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and the like.

[0085] The term “heteroaliphatic” as used herein refers to aliphaticmoieties which contain one or more oxygen, sulfur, or nitrogen atoms,e.g., in place of carbon atoms.

[0086] The term “heterocycle” as used herein refers to cyclic aliphaticgroups having one or more heteroatoms, and preferably three to sevenring atoms total, includes, but is not limited to oxetane,tetrahydrofuranyl, tetrahydropyranyl, aziridine, azetidine, pyrrolidine,piperidine, morpholine, piperazine and the like.

[0087] The terms “aryl” and “heteroaryl” as used herein refer to stablemono- or polycyclic, heterocyclic, polycyclic, and polyheterocyclicunsaturated moieties having 3-14 carbon atom which may be substituted orunsubstituted. Non-limiting examples of useful aryl ring groups includephenyl, halophenyl, alkoxypheriyl, dialkoxyphenyl, trialkoxyphenyl,alkylenedioxyphenyl, naphthyl, phenanthryl, anthryl, phenanthro and thelike. Examples of typical heteroaryl rings include 5-membered monocyclicring groups such as thienyl, pyrrolyl, imidazolyl, pyrazolyl, furyl,isothiazolyl, furazanyl, isoxazolyl, thiazolyl and the like; 6-memberedmonocyclic groups such as pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,triazinyl and the like; and polycyclic heterocyclic ring groups such asbenzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, isobenzofuranyl,chromenyl, xanthenyl, phenoxathienyl, indolizinyl, isoindolyl, indolyl,indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl,quinoxalinyl, quinazolinyl, benzothiazole, benzimidazole,tetrahydroquinoline cinnolinyl, pteridinyl, carbazolyl, beta-carbolinyl,phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl,isothiazolyl, phenothiazinyl, phenoxazinyl, and the like(see e.g.Katritzky, Handbook of Heterocyclic Chemistry). The aryl or heteroarylmoieties may be substituted with one to five members selected from thegroup consisting of hydroxy, C1-C8 alkoxy, C1-C8 branched orstraight-chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro,halo, trihalomethyl, cyano, and carboxyl.

[0088] A “halo” substituent according to the present invention may be afluoro, chloro, bromo or iodo substituent.

[0089] As discussed above, R¹ may be aliphatic, heteroaliphatic, aryl orheteroaryl and usually comprises one to about 36 carbon atoms, exclusiveof optional substituents.

[0090] In certain embodiments, R¹ is optionally be joined, i.e.,covalently linked, to R², B¹ or B², forming a macrocyclic structure.

[0091] In certain embodiments —XR¹ is a moiety of the formula

[0092] where R⁴ is a H, aliphatic, heteroaliphatic, aryl or heteroaryl.The aliphatic moieties may be branched, unbranched, cyclic, saturated orunsaturated, substituted or unsubstituted and include, e.g, methyl,ethyl, isopropyl, t-butyl, cyclopentyl, cyclohexyl, etc. Heteroaliphaticmoieties may be branched, unbranched or cyclic and include heterocyclessuch as morpholino, pyrrolidinyl, etc. Illustrative ortho-, meta- orpara-, substitutents for a phenyl group at this position include one ormore of the following: halo, e.g. chloro or flouro; hydroxyl, amino,—SO₂NH₂, —SO₂NH(aliphatic), —SO₂N(aliphatic)₂, —O-aliphatic-COOH,—O-aliphatic-NH₂ (which may contain one or two N-aliphatic or N-acylsubstituents), C1-C6 alkyl, acyl, acyloxy, C1-C6 alkoxy, e.g. methoxy,ethoxy, methylenedioxy, ethylenedioxy, etc. Heteroaryl groups are asdiscussed previously, including indolyl, pyridyl, pyrrolyl, etc.Particular R⁴ moieties include the following:

[0093] R⁵ is a branched, unbranched or cyclic aliphatic moiety of 1 to 8carbon atoms, which may be optionally substituted, including forexample, —CH—, —CHCH2—, —CH₂CH—, —CHCH₂CH₂₋—, —CH₂CHCH₂—,—CH(CH₃)—CH₂—CH, —CH(CH₂CH₃)—CH₂—CH, —CH₂CH₂CH—, —C(CH₃)CH₂—, and thelike;

[0094] R⁶ is an aliphatic, heteroaliphatic, heterocylic, aryl orheteroaryl moiety, which may be substituted or unsubstituted. Typicalsubstituents for R⁶ include branched, unbranched or cyclic, C1-C8,aliphatic or heteroaliphatic groups, including unsaturated groups suchas substitute or unsubstituted alkenes, heterocycles, phenyl, etc.

[0095] R⁷ is H or a substituent such as, in certain embodiments,—(CH₂)_(z)—CH═CH₂, —(CH₂)_(z)—COOH, —(CH₂)_(z)—CHO, —(CH₂)_(z)—OH,—(CH₂)_(z)—NH₂, —(CH₂)_(z)—NH—alkyl, —(CH₂)_(z)—SH, or an amino groupwhich may be substituted or unsubstituted (preferably a tertiary amine),etc. In embodiments where R⁶ is aryl, R⁷ may be present in the o, m, orp position. z is an integer from 0 through 4.

[0096] As discussed above, B¹, B² and R² may be aliphatic,heteroaliphatic, aryl or heteroaryl. Typical groups include a branched,unbranched or cyclic, saturated or unsaturated, aliphatic moiety,preferably of 1 to about 12 carbon atoms (including for example methyl,ethyl, n-propyl, isopropyl, cyclopropyl, —CH₂-cyclopropyl, allyl,n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, —CH₂-cyclobutyl,n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl,—CH₂—cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, —CH₂-cyclohexyl andthe like), which aliphatic moiety may optionally be substituted with an—OH, —C═O, —COOH, CHO, allyl, NH₂ (or substituted amine, amide, urea orcarbamate), ether (or thio-ether, in either case, aliphatic oraromatic), aryl, or heteroaryl moiety, and may optionally contain aheteroatom in place of one or more CH₂ or CH units; or a substituted orunsubstituted aryl (e.g. mono-, di- and tri-alkoxyphenyl;methylenedioxyphenyl or ethylenedioxyphenyl; halophenyl; or-phenyl-C(Me)₂—CH₂—O—CO—[C3-C6] alkyl or alkylamino) or heteroaromaticmoiety. In such embodiments, where YR² is -OPhenyl and B¹ is H, B² ispreferably not cyclopentyl. In other embodiments, Y is NH and the moiety—(C═O)—CH(B¹)NHR² comprises among other groups, D- or L-forms ofnaturally occurring or synthetic alpha amino acids as well as N-alkyl,N-acyl, N-aryl and N-aroyl derivatives thereof. Particular XR¹, G, B¹,B² and YR² groups for the various foregoing structures further includethose illustrated in compounds described in the examples, tables ofmonomers and dimers and other disclosure in WO 96/06097, WO 97/31899 andWO 97/31898.

[0097] One preferred class of compounds are those compounds of Genus Iin which n is 2.

[0098] Another preferred class of compounds are those compounds of GenusI in which B¹ is H; B² is branched, unbranched or cyclic, saturated orunsaturated, aliphatic moiety, preferably of 1 to 8, more preferably 1to 6, carbon atoms (including for example methyl, ethyl, n-propyl,isopropyl, cyclopropyl, —CH₂-cyclopropyl, allyl, n-butyl, sec-butyl,isobutyl, tert-butyl, cyclobutyl, —CH₂-cyclobutyl, n-pentyl, sec-pentyl,isopentyl, tert-pentyl, cyclopentyl, —CH₂-cyclopentyl, n-hexyl,sec-hexyl, cyclohexyl, —CH₂-cyclohexyl and the like), which aliphaticmoiety may optionally be substituted, e.g. with an —OH, —C═O, —COOH,CHO, allyl, NH₂ (or substituted amine, amide, urea or carbamate), orether (or thio-ether, in either case, aliphatic or aromatic), and mayoptionally contain a heteroatom in place of one or more CH₂ or CH units;and YR² is aryl, heteroaryl and may be optionally substituted (YR², forinstance, includes moieties such as o-, m-, or p-alkoxyphenyl; 3,5-,2,3-, 2,4-, 2,5-, 3,4- or 3,5-dialkoxyphenyl, or 3,4,5-trialkoxyphenyl,e.g. where the alkoxy groups are independently selected from methoxy andethoxy (one or more of which may bear a hydroxy or amino moiety).

[0099] Another preferred class of compounds are those compounds of GenusI in which B¹, B² and YR² are the same or different lower aliphaticmoieties.

[0100] Another preferred class of compounds are those compounds of GenusI which contain a moiety —NB¹R² in which B¹ is H and R² is loweraliphatic.

[0101] Another preferred class of compound are those compounds of GenusI in which G is an alicyclic or heterocyclic group bearing optionalsubstituents.

[0102] Another preferred class of compounds are those compounds of GenusI in which X is oxygen and R¹ comprises R⁴R⁵R⁶R⁷ where R⁴ is aliphatic,alicyclic, aryl, heteroaryl, or heterocyclic, optionally substituted; R⁵is a branched or unbranched lower aliphatic group; R⁶ is aliphatic,alicyclic, heteroaliphatic, heterocyclic, aryl or heteroaryl, optionallysubstituted.

[0103] Another preferred class of compounds are those compounds of GenusI in which R1 comprises R⁴R⁵R⁶R⁷ as described in the immediatelypreceding paragraph and YR²comprises a substituted or unsubstituted arylor heteroaryl, including phenyl; o-, m- or p- substituted phenyl wherethe substituent is halo such as chloro, lower alkyl, or alkoxy, such asmethoxy or ethoxy; disubstituted phenyl, e.g. dialkoxyphenyl such as2,4-, 3,4- or 3.5-dimethoxy or diethoxy phenyl or such asmethylenedioxyphenyl, or 3-methoxy-5-ethoxyphenyl; or trisubstitutedphenyl, such as trialkoxy (e.g., 3,4,5-trimethoxy or ethoxyphenyl),3,5-dimethoxy-4-chloro-phenyl, etc.).

[0104] In addition, such compounds may comprise a substituted prolineand pipecolic acid derivative, numerous examples of which have beendescribed in the literature. Using synthetic procedures similar to thosedescribed in the patent documents and scientific literature citedherein, substituted prolines and pipecolates can be utilized to prepareligands with substituents at positions C-2 to C-6 (with reference to theFK506 numbering of most of the references cited below), as exemplifiedin the patent applications cited herein.

[0105] For representative examples of substituted prolines and pipecolicacids see: Chung, et al., J. Org. Chem., 1990, 55, 270; Shuman, et al.,J. Org. Chem., 1990, 55, 738; Hanson, et al., Tetrahedron Lett., 1989,30, 5751; Bailey, et al., Tetrahedron Lett., 1989, 30, 6781.

[0106] For a variety of guidance on chemical transformations, synthesis,formulation and delivery of a variety of compounds, including additionalinformation relating to FKBP ligands and/or to ligands for other ligandbinding domains, see e.g., WO 94/18317 and Belshaw et al, 1996, PNAS93:4604-4607) (for methods and materials based on ligands for animmunophilin such as FKBP, a cyclophilin, and/or FRB domain); WO96/06097 and WO 97/31898 (more ligands for FKBP and variants thereof);WO 93/33052, WO 96/41865 and Rivera et al, “A humanized system forpharmacologic control of gene expression”, Nature Medicine2(9):1028-1032 (1997)) (rapamycin analogs); WO 94/18317(cyclophilin/cyclosporin); Licitra et al, 1996, Proc. Natl. Acad. Sci.USA 93:12817-12821 (DHFR/methotrexate); and Farrar et al, 1996, Nature383:178-181 (DNA gyrase/coumermycin). Numerous variations andmodifications to ligands and ligand binding domains, as well asmethodologies for designing, selecting and/or characterizing them, whichmay be adapted to the present invention are disclosed in the citedreferences.

[0107] Cleavage Enzymes:

[0108] It is often preferable in the design of fusion proteins of thisinvention to have an enzymatic cleavage site located between the CRD andthe target protein. When the fusion protein exits the ER followingaddition of ligand, the enzymatic cleavage site allows the targetprotein to be released from the CRD and secreted. Ideally, the cleavagesite should be specific to an enzyme which resides in a cellularcompartment between the ER and the plasma membrane, e.g. the Golgiapparatus. An exemplary cleavage enzyme is furin, also known as PACE.Furin is a member of the KEX2/subtilisin family of pro-proteinconvertases, which convert pro-proteins and pro-hormones to their activeforms (Kazuhisa Nakayama, Biochem J. (1997) 327:625-635). It is aprotein which resides in the trans-golgi, although like many golgiproteins such as TGN38, it constitutively cycles between the cellsurface and the TGN (trans-golgi network). Furin has a ubiquitous tissuedistribution and its substrates are numerous and varied. However, nearlyall share the consensus cleavage sequence RX(K/R)R. Proteins which aresubstrates for furin include: human pro-neurotrophin-3 (MSMRVRR), humanpro-insulin like growth factor I (KPAKSAR), human pro-parathyroidhormone (KSVKKR), human stromelysin-3 (ARNRQKR). Furin is also capableof cleaving membrane bound substrates, such as human insulinpro-receptor (RPSRKRR) and human hepatocyte growth factor pro-receptor(TEKRKKR). A cleavage site from any furin substrate can be used in thefusion proteins of the invention. In some cases, the site will be be anon-naturally occurring peptide sequence containing the consensus furincleavage sequence. One particular advantage of having furin as thecleavage enzyme is that its recognition sequence is located exclusivelyN-terminal to the cleavage site. This allows the portion of the proteinthat encodes the target protein to be released from the cell unalteredby the presence of additional amino acids.

[0109] The furin family contains other members which may also be usefulin the practice of this invention. Many of these proteins have a uniquetissue distribution. For example, PC1/PC3 and PC2 are only found inneuroendocrine tissues like pancreatic islets, pituitary and brain andPC4 is expressed primarily within testicular-germ cells. PACE4, as wellas PC5/PC6 and LPC/PC7/PC8/SPC7 are expressed ubiquitously (Nakayama,1997). Cleavage sites for these enzymes may also be used in the practiceof this invention, provided the fusion proteins are expressed in theappropriate cell type.

[0110] In addition, any mammalian protease with a specific cleavagesequence, such as subtilisin, could be used to cleave the fusionproteins of this invention, if it were targeted to the desired locationin the cell. For example, subtilisin could be targeted to the TGN byfusing it to a localization sequence from a resident golgi protein suchas TGN38. Alternatively, the motifs which are known to target furin tothe TGN, including YKGL and the Ser-containing cluster SDSEEDE, maysuffice to target a cytoplasmic protease to the Golgi. Cells may also beengineered to express an enzyme tailored to cut a sequence found only inthe CRD containing fusion protein. For example, Ballinger et al.describe mutant forms of sub tilisin in which the enzyme has beenengineered to acquire the specificity of furin (Ballinger Md., et al.Biochemistry. Oct 22, 1996;35(42):13579-85. Ballinger Md., et al.Biochemistry. Oct 17, 1995;34(41):13312

[0111] Secretory Signal Sequences:

[0112] When secretory proteins are translated on the ribosome, an aminoacid sequence of 16-30 residues, known as the signal sequence, directsthe ribosome to the ER membrane. This sequence then initiates a signalwhich transports the nascent chain into the ER, across the ER membrane.Generally, such sequences are found at the N-terminus of a protein andcontain one or more positively charged amino acids followed by a stretchof 6-12 hydrophobic residues. Numerous signal sequences are known, andany signal sequence which normally directs the translocation of asecretory or transmembrane protein to the ER may be used in the fusionproteins of this invention. Exemplary signal sequences are those frompreproalbumin, prelysozyme, human growth hormone, proinsulin,acetylcholine receptor or IgG light chain. For use in this invention, asignal sequence is encoded at the N-terminus of the protein to beregulatably secreted. This signal sequence then directs the ribosome tothe ER, where the translated protein containing the CRD aggregates untilligand is added to the cell.

[0113] Target Proteins:

[0114] Fusion proteins of this invention may contain any target proteinwhich one may want to secrete or translocate rapidly and efficiently.Preferably, the target protein will be a therapeutic protein. The targetprotein can provide a desired phenotype. It can be a membrane-bound ormembrane-spanning protein, a secreted protein, or a cytoplasmic protein.The proteins which are expressed, singly or in combination, can involvehoming, cytotoxicity, proliferation, differentiation, immune response,inflammatory response, clotting, thrombolysis, hormonal regulation,angiogenesis, etc. The polypeptide may be of naturally occurring ornon-naturally occurring peptide sequence.

[0115] Various secreted products include hormones, such as insulin,human growth hormone, glucagon, pituitary releasing factor, ACTH,melanotropin, relaxin, leptin, etc.; growth factors, such as EGF, IGF-1,TGF-alpha, -beta, PDGF, G-CSF, M-CSF, GM-CSF, members of the FGF family,erythropoietin, thrombopoietin, megakaryocytic growth factors, nervegrowth factors, etc.; proteins which stimulate or inhibit angiogenesissuch as angiostatin, endostatin and VEGF and variants thereof;interleukins, such as IL-1 to -15; TNF-alpha and -beta; interferons-alpha, -beta and -gamma; and enzymes and other factors, such as tissueplasminogen activator, members of the complement cascade, perforins,superoxide dismutase; coagulation-related factors such asantithrombin-III, Factor V, Factor VII, Factor VIIIc, vWF, Factor IX,alpha-anti-trypsin, protein C, and protein S; endorphins, dynorphin,bone morphogenetic protein, CFTR, etc.

[0116] The protein may be a naturally-occurring surface membrane proteinor a protein made so by introduction of an appropriate signal peptideand transmembrane sequence. Various such proteins include homingreceptors, e.g. L-selectin (Mel-14), hematopoietic cell markers, e.g.CD3, CD4, CD8, B cell receptor, TCR subunits alpha, beta, gamma ordelta, CD10, CD19, CD28, CD33, CD38, CD41, etc., receptors, such as theinterleukin receptors IL-2R, IL-4R, etc.; receptors for other ligandsincluding the various hormones, growth factors, etc.; receptorantagonists for such receptors and soluble forms of such receptors;channel proteins, for influx or efflux of ions, e.g. H⁺, Ca⁺², K⁺, Na⁺,Cl⁻, etc., and the like; CFTR, tyrosine activation motif, zap-70, etc.

[0117] The target protein can be an intracellular protein such as aprotein involved in a metabolic pathway, or a regulatory protein,steroid receptor, transcription factor, etc.,

[0118] By way of further illustration, in T-cells, one may wish tointroduce genes encoding one or both chains of a T-cell receptor. ForB-cells, one could provide the heavy and light chains for animmunoglobulin for secretion. For cutaneous cells, e.g. keratinocytes,particularly keratinocyte stem cells, one could provide for protectionagainst infection, by secreting alpha, beta or gamma interferon,antichemotactic factors, proteases specific for bacterial cell wallproteins, various anti-viral proteins, etc.

[0119] In various situations, one may wish to direct a cell to aparticular site. The site can include anatomical sites, such as lymphnodes, mucosal tissue, skin, synovium, lung or other internal organs orfunctional sites, such as clots, injured sites, sites of surgicalmanipulation, inflammation, infection, etc. Regulated expression of amembrane protein which recognizes or binds to the particular site ofinterest, for example, provides a method for directing the engineeredcells to that site. Thus one can achieve a localized concentration of asecreted product or effect cell-based healing, scavenging, protectionfrom infection, anti-tumor activity, etc. Proteins of interest includehoming receptors, e.g. L-selectin, GMP140, CLAM-1, etc., or addressins,e.g. ELAM-1, PNAd, LNAd, etc., clot binding proteins, or cell surfaceproteins that respond to localized gradients of chemotactic factors.

[0120] In one embodiment of this invention, binding of a ligand to a CRDregulates transcription of a target gene. In this embodiment, the targetgene may encode any protein, including those described above.

[0121] Disposal Targeting Sequences:

[0122] In many embodiments of the invention, it would be desirable todispose of the CRD following its cleavage from the target protein.Disposal of the CRD would prevent its secretion from the cell and itsaccumulation in the bloodstream. One way to achieve this goal is totarget the CRD to a lysosomal compartment, where it would be degraded.During normal cellular trafficking, lysosomal proteins are sorted fromthe trans-golgi network, where they are directed to the endosomalpathway, and subsequently, to lysosomes. Resident soluble lysosomalenzymes such as cathepsin D are marked for targeting to the lysosomalpathway by attachment of a phosphate group on carbon 6 of one or moremannose residues on a particular N-linked oligosaccharide, which arethen recognized by the mannose-6-phosphate receptor in the lysozyme. Thephosphotransferase recognition sequence of cathepsin D consists of twodiscontinuous sequences: amino acids 188-230, including a criticallysine residue at position 203, and amino acids 265-292 (Baranski etal., Cell 1990, 63:281-291.) Baranski et al. have demonstrated thatsplicing of these sequences into the appropriate location on pepsinogen,a secretory protein, resulted in phosphorylation of the sugars on thechimeric molecule (Baranski et al., supra). Other groups have shown thatfusion of the entire cathepsin B sequence onto MyoD resulted intargeting of the complex to the lysosome (Li et al., J. Cell Biol.,135:1043-1057, November 1996.) Similarly, chimeric proteins consistingof soluble CD4, procathepsin D and the C-terminal tails of threelysosomal membrane proteins were able to direct the HIV glycoprotein gp160 to the lysosome for degradation (Lin et al., FASEB J., 7:1070-1080,August 1993.) Lysosomal membrane proteins such as lamp-1 and LAP aredirected to the lysosome via a tyrosine-based targeting motif in theirC-terminal tails (Williams et al., J. Cell Biol., 111:955-966, 1990;Klionsky et al., J. Biol. Chem., 265:5349-5352, 1990.) Fusion of thesetails onto the extracellular and transmembrane domains of residentplasma membrane proteins is sufficient to target those proteins to thelysosome.

[0123] Either of the aforementioned lysosomal targeting signals may beused to target CRDs of this invention for disposal. For solubleproteins, the preferred method is to fuse a resident lysosomal proteincontaining a mannose-6-phosphate signal to the CRD. Examples of suchproteins are the cysteine proteases of the cathepsin family: cathepsinsB, D, H, L, S, C and K. Other lysosomal enzymes which may be usedinclude the carboxypeptidases prolylcarboxypeptidase and deamidase(cathepsin A). For membrane bound CRDs, the preferred targeting sequencewould be one found in lysosomal membrane proteins, e.g. a tyrosine-basedinternalization motif. These motifs are short, linear stretches of aminoacids within the cytoplasmic region of the protein to be targeted.Tyrosine-based motifs center on a critical tyrosine residue within thesequence NPXY or YXXØ, where X is any amino acid and Ø is an amino acidwith a bulky hydrophobic group. In many proteins, a glycine precedingthe tyrosine in a YXXØ-type signal enhances targeting of these proteinsto the lysosome. Sequences for use in some applications of thisinvention may be derived from proteins such as Lamp-1, LAP (lysosomalacid phosphatase), CD63, Lamp-2 or CD3-gamma, all of which are normallytargeted to the lysosome. For additional information on tyrosine-basedsorting motifs, see, for example the review by Marks et al., Trends inCell Biology, 7:124-128, 1997.

[0124] Design and Assembly of the DNA Constructs

[0125] Constructs may be designed in accordance with the principles,illustrative examples and materials and methods disclosed in the patentdocuments and scientific literature cited herein, with modifications andfurther exemplification as described. Components of the constructs canbe prepared in conventional ways, where the coding sequences andregulatory regions may be isolated, as appropriate, ligated, cloned inan appropriate cloning host, analyzed by restriction or sequencing, orother convenient means. Particularly, using PCR, individual fragmentsincluding all or portions of a functional unit may be isolated, whereone or more mutations may be introduced using “primer repair”, ligation,in vitro mutagenesis, etc. as appropriate. In the case of DNA constructsencoding fusion proteins, DNA sequences encoding individual domains andsub-domains are joined such that they constitute a single open readingframe encoding a fusion protein capable of being translated in cells orcell lysates into a single polypeptide harboring all component domains.The DNA construct encoding the fusion protein may then be placed into avector for transducing host cells and permitting the expression of theprotein. For biochemical analysis of the encoded chimera, it may bedesirable to construct plasmids that direct the expression of theprotein in bacteria or in reticulocyte-lysate systems. For use in theproduction of proteins in mammalian cells, the protein-encoding sequenceis introduced into an expression vector that directs expression in thesecells. Expression vectors suitable for such uses are well known in theart. Various sorts of such vectors are commercially available.

[0126] Promoters

[0127] The fusion proteins described herein may be used in combinationwith any promoter that will direct their expression in mammalian cells.The promoter may be a strong promoter, such as the human CMV promoter,or a weaker promoter, such as a promoter for an endogenous human gene.Other promoters which may be used include, but are not limited to, theRous Sarcoma Virus (RSV) promoter, the retroviral LTR from MurineMoloney Leukemia Virus (MMLV), the muscle creatine kinase (MCK)enhancer, the SV40 promoter, and the CMV enhancer from the majorimmediate early gene. Genbank accession numbers for the above promotersare given in the table below. Promoter Genbank Accession Number CMVAF067197 RSV M83237 MMLV LTR M77239 SV40 U47120 CMV enhancer for MIEgene K03104 MCK enhancer X67536

[0128] In many cases, the selection of promoter will depend upon theconfiguration of the fusion protein used in a particular application.Thus, if the practitioner desired the CRD-containing fusion protein tobe expressed at high levels, a stronger promoter, such as CMV, would beused. Alternatively, for tissue specific expression, a tissue specificpromoter like the MCK enhancer (for expression in muscle) would beselected.

[0129] Introduction of Constructs into Cells

[0130] This invention is particularly useful for the engineering ofanimal cells and in applications involving the use of such engineeredanimal cells. The animal cells may be, among others, insect, worm ormammalian cells. While various mammalian cells may be used, including,by way of example, equine, bovine, ovine, canine, feline, murine, andnon-human primate cells, human and mouse cells are of particularinterest. Across the various species, various types of cells may beused, such as hematopoietic, neural, glial, mesenchymal, cutaneous,mucosal, stromal, muscle (including smooth muscle cells), spleen,reticuloendothelal, epithelial, endothelial, hepatic, kidney,gastrointestinal, pulmonary, fibroblast, and other cell types. Ofparticular interest are muscle cells (including skeletal, cardiac andother muscle cells), cells of the central and peripheral nervoussystems, and hematopoietic cells, which may include any of the nucleatedcells which may be involved with the erythroid, lymphoid ormyelomonocytic lineages, as well as myoblasts and fibroblasts. Also ofinterest are stem and progenitor cells, such as hematopoietic, neural,stromal, muscle, hepatic, pulmonary, gastrointestinal and mesenchymalstem cells

[0131] The cells may be autologous cells, syngeneic cells, allogeneiccells and even in some cases, xenogeneic cells with respect to anintended host organism. The cells may be modified by changing the majorhistocompatibility complex (“MHC”) profile, by inactivatingβ2-microglobulin to prevent the formation of functional Class I MHCmolecules, inactivation of Class II molecules, providing for expressionof one or more MHC molecules, enhancing or inactivating cytotoxiccapabilities by enhancing or inhibiting the expression of genesassociated with the cytotoxic activity, and the like.

[0132] In some instances specific clones or oligoclonal cells may be ofinterest, where the cells have a particular specificity, such as T cellsand B cells having a specific antigen specificity or homing target sitespecificity.

[0133] Constructs encoding the fusion proteins and comprising targetgenes of this invention can be introduced into the cells as one or morenucleic acid molecules or constructs, in many cases in association withone or more markers to allow for selection of host cells which containthe construct(s). The constructs can be prepared in conventional ways,where the coding sequences and regulatory regions may be isolated, asappropriate, ligated, cloned in an appropriate cloning host, analyzed byrestriction or sequencing, or other convenient means. Particularly,using PCR, individual fragments including all or portions of afunctional domain may be isolated, where one or more mutations may beintroduced using “primer repair”, ligation, in vitro mutagenesis, etc.as appropriate.

[0134] The construct(s) once completed and demonstrated to have theappropriate sequences may then be introduced into a host cell by anyconvenient means. The constructs may be incorporated into vectorscapable of episomal replication (e.g. BPV or EBV vectors) or intovectors designed for integration into the host cells' chromosomes. Theconstructs may be integrated and packaged into non-replicating,defective viral genomes like Adenovirus, Adeno-associated virus (AAV),or Herpes simplex virus (HSV) or others, including retroviral vectors,for infection or transduction into cells. Alternatively, the constructmay be introduced by protoplast fusion, electroporation, biolistics,calcium phosphate transfection, lipofection, microinjection of DNA orthe like. The host cells will in some cases be grown and expanded inculture before introduction of the construct(s), followed by theappropriate treatment for introduction of the construct(s) andintegration of the construct(s). The cells may then be expanded and/orscreened by virtue of a marker present in the constructs. Variousmarkers which may be used successfully include hprt, neomycinresistance, thymidine kinase, hygromycin resistance, etc., and variouscell-surface markers such as Tac, CD8, CD3, Thy1 and the NGF receptor.

[0135] In some instances, one may have a target site for homologousrecombination, where it is desired that a construct be integrated at aparticular locus. For example, one can delete and/or replace anendogenous gene (at the same locus or elsewhere) with a recombinanttarget construct of this invention. For homologous recombination, onemay generally use either Ω or O-vectors. See, for example, Thomas andCapecchi, Cell (1987) 51, 503-512; Mansour, et al., Nature (1988) 336,348-352; and Joyner, et al., Nature (1989) 338, 153-156.

[0136] The constructs may be introduced as a single DNA moleculeencoding all of the genes, or different DNA molecules having one or moregenes. The constructs may be introduced simultaneously or consecutively,each with the same or different markers.

[0137] Vectors containing useful elements such as bacterial or yeastorigins of replication, selectable and/or amplifiable markers,promoter/enhancer elements for expression in prokaryotes or eukaryotes,and mammalian expression control elements, etc. which may be used toprepare stocks of construct DNAs and for carrying out transfections arewell known in the art, and many are commercially available.

[0138] Introduction of Constructs into Animals

[0139] Any means for the introduction of genetically engineered cells orheterologous DNA into animals, preferably mammals, human or non-human,may be adapted to the practice of this invention for the delivery of thevarious DNA constructs into the intended recipient. For the purpose ofthis discussion, the various DNA constructs described herein maytogether be referred to as the transgene.

[0140] By ex vivo Genetic Engineering

[0141] Cells which have been transduced ex vivo or in vitro with the DNAconstructs may be grown in culture under selective conditions and cellswhich are selected as having the desired construct(s) may then beexpanded and further analyzed, using, for example, the polymerase chainreaction for determining the presence of the construct in the host cellsand/or assays for the production of the desired gene product(s). Afterbeing transduced with the heterologous genetic constructs, the modifiedhost cells may be identified, selected, grown, characterized, etc. asdesired, and then may be used as planned, e.g. grown in culture orintroduced into a host organism.

[0142] Depending upon the nature of the cells, the cells may beintroduced into a host organism, e.g. a mammal, in a wide variety ofways, generally by injection or implantation into the desired tissue orcompartment, or a tissue or compartment permitting migration of thecells to their intended destination. Illustrative sites for injection orimplantation include the vascular system, bone marrow, muscle, liver,cranium or spinal cord, peritoneum, and skin. Hematopoietic cells, forexample, may be administered by injection into the vascular system,there being usually at least about 10⁴ cells and generally not more thanabout 10¹⁰ cells. The number of cells which are employed will dependupon the circumstances, the purpose for the introduction, the lifetimeof the cells, the protocol to be used, for example, the number ofadministrations, the ability of the cells to multiply, the stability ofthe therapeutic agent, the physiologic need for the therapeutic agent,and the like. Generally, for myoblasts or fibroblasts for example, thenumber of cells will be at least about 10⁴ and not more than about 10⁹and may be applied as a dispersion, generally being injected at or nearthe site of interest. The cells will usually be in aphysiologically-acceptable medium.

[0143] Cells engineered in accordance with this invention may also beencapsulated, e.g. using conventional biocompatible materials andmethods, prior to implantation into the host organism or patient for theproduction of a therapeutic protein. See e.g. Hguyen et al, TissueImplant Systems and Methods for Sustaining viable High Cell Densitieswithin a Host, U.S. Pat. No. 5,314,471 (Baxter International, Inc.);Uludag and Sefton, 1993, J Biomed. Mater. Res. 27(10):1213-24 (HepG2cells/hydroxyethyl methacrylate-methyl methacrylate membranes); Chang etal, 1993, Hum Gene Ther 4(4):433-40 (mouse Ltk- cells expressinghGH/immunoprotective perm-selective alginate microcapsules; Reddy et al,1993, J Infect Dis 168(4):1082-3 (alginate); Tai and Sun, 1993, FASEB J7(11):1061-9 (mouse fibroblasts expressinghGH/alginate-poly-L-lysine-alginate membrane); Ao et al, 1995,Transplantation Proc. 27(6):3349, 3350 (alginate); Rajotte et al, 1995,Transplantation Proc. 27(6):3389 (alginate); Lakey et al, 1995,Transplantation Proc. 27(6):3266 (alginate); Korbutt et al, 1995,Transplantation Proc. 27(6):3212 (alginate); Dorian et al, U.S. Pat. No.5,429,821 (alginate); Emerich et al, 1993, Exp Neurol 122(1):37-47(polymer-encapsulated PC12 cells); Sagen et al, 1993, J Neurosci13(6):2415-23 (bovine chromaffin cells encapsulated in semipermeablepolymer membrane and implanted into rat spinal subarachnoid space);Aebischer et al, 1994, Exp Neurol 126(2):151-8 (polymer-encapsulated ratPC12 cells implanted into monkeys; see also Aebischer, WO 92/19595);Savelkoul et al, 1994, J Immunol Methods 170(2):185-96 (encapsulatedhybridomas producing antibodies; encapsulated transfected cell linesexpressing various cytokines); Winn et al, 1994, PNAS USA 91(6):2324-8(engineered BHK cells expressing human nerve growth factor encapsulatedin an immunoisolation polymeric device and transplanted into rats);Emerich et al, 1994, Prog Neuropsychopharmacol Biol Psychiatry18(5):935-46 (polymer-encapsulated PC12 cells implanted into rats);Kordower et al, 1994, PNAS USA 91(23):10898-902 (polymer-encapsulatedengineered BHK cells expressing hNGF implanted into monkeys) and Butleret al WO 95/04521 (encapsulated device). The cells may then beintroduced in encapsulated form into an animal host, preferably a mammaland more preferably a human subject in need thereof. Preferably theencapsulating material is semipermeable, permitting release into thehost of secreted proteins produced by the encapsulated cells. In manyembodiments the semipermeable encapsulation renders the encapsulatedcells immunologically isolated from the host organism in which theencapsulated cells are introduced. In those embodiments the cells to beencapsulated may express one or more fusion proteins containingcomponent domains derived from proteins of the host species and/or fromviral proteins or proteins from species other than the host species. Thecells may be derived from one or more individuals other than therecipient and may be derived from a species other than that of therecipient organism or patient.

[0144] By in vivo Genetic Engineering

[0145] Instead of ex vivo modification of the cells, in many situationsone may wish to modify cells in vivo. A variety of techniques have beendeveloped for genetic engineering of target tissue and cells in vivo,including viral and non-viral systems.

[0146] In one approach, the DNA constructs are delivered to cells bytransfection, i.e., by delivery to cells of “naked DNA”, lipid-complexedor liposome-formulated DNA, or otherwise formulated DNA. Prior toformulation of DNA, e.g., with lipid, or as in other approaches, priorto incorporation in a final expression vector, a plasmid containing atransgene bearing the desired DNA constructs may first be experimentallyoptimized for expression (e.g., inclusion of an intron in the 5′untranslated region and elimination of unnecessary sequences (Felgner,et al., Ann NY Acad Sci 126-139, 1995). Formulation of DNA, e.g. withvarious lipid or liposome materials, may then be effected using knownmethods and materials and delivered to the recipient mammal. See, e.g.,Canonico et al, Am J Respir Cell Mol Biol 10:24-29, 1994 (in vivotransfer of an aerosolized recombinant human alphal-antitrypsin genecomplexed to cationic liposomes to the lungs of rabbits); Tsan et al, AmJ Physiol 268 (Lung Cell Mol Physiol 12): L1052-L1056, 1995 (transfer ofgenes to rat lungs via tracheal insufflation of plasmid DNA alone orcomplexed with cationic liposomes); Alton et al., Nat Genet. 5:135-142,1993 (gene transfer to mouse airways by nebulized delivery ofcDNA-liposome complexes). In either case, delivery of vectors or nakedor formulated DNA can be carried out by instillation via bronchoscopy,after transfer of viral particles to Ringer's, phosphate bufferedsaline, or other similar vehicle, or by nebulization.

[0147] Viral systems include those based on viruses such as adenovirus,adeno-associated virus, hybrid adeno-AAV, lentivirus and retroviruses,which allow for transduction by infection, and in some cases,integration of the virus or transgene into the host genome. See, forexample, Dubensky et al. (1984) Proc. Natl. Acad. Sci. USA 81,7529-7533; Kaneda et al., (1989) Science 243,375-378; Hiebert et al.(1989) Proc. Natl. Acad. Sci. USA 86, 3594-3598; Hatzoglu et al. (1990)J. Biol. Chem. 265, 17285-17293 and Ferry, et al. (1991) Proc. Natl.Acad. Sci. USA 88, 8377-8381. The virus may be administered by injection(e.g. intravascularly or intramuscularly), inhalation, or otherparenteral mode. Non-viral delivery methods such as administration ofthe DNA via complexes with liposomes or by injection, catheter orbiolistics may also be used. See e.g. WO 96/41865, PCT/US97/22454 andU.S. Ser. No. 60/084819, for example, for additional guidance onformulation and delivery of recombinant nucleic acids to cells and toorganisms.

[0148] By employing an attenuated or modified retrovirus carrying atarget transcriptional initiation region, if desired, one can activatethe virus using one of the subject transcription factor constructs, sothat the virus may be produced and transduce adjacent cells.

[0149] The use of recombinant viruses to deliver the nucleic acidconstructs are of particular interest. The transgene(s) may beincorporated into any of a variety of viruses useful in gene therapy.

[0150] In clinical settings, the gene delivery systems (i.e., therecombinant nucleic acids in vectors, virus, lipid formulation or otherform) can be introduced into a patient, e.g., by any of a number ofknown methods. For instance, a pharmaceutical preparation of the genedelivery system can be introduced systemically, e.g. by intravenousinjection, inhalation, etc. In some systems, the means of deliveryprovides for specific or selective transduction of the construct intodesired target cells. This can be achieved by regional or localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection, e.g. Chen et al., (1994) PNAS USA 91: 3054-3057 or bydeterminants of the delivery means. For instance, some viral systemshave a tissue or cell-type specificity for infection. In some systemscell-type or tissue-type expression is achieved by the use of cell-typeor tissue-specific expression control elements controlling expression ofthe gene.

[0151] In preferred embodiments of the invention, the subject expressionconstructs are derived by incorporation of the genetic construct(s) ofinterest into viral delivery systems including a recombinant retrovirus,adenovirus, adeno-associated virus (AAV), hybrid adenovirus/AAV, herpesvirus or lentivirus (although other applications may be carried outusing recombinant bacterial or eukaryotic plasmids). While various viralvectors may be used in the practice of this invention, AAV- andadenovirus-based approaches are of particular interest for the transferof exogenous genes in vivo, particularly into humans and other mammals.The following additional guidance on the choice and use of viral vectorsmay be helpful to the practitioner, especially with respect toapplications involving whole animals (including both human gene therapyand the development and use of animal model systems), whether ex vivo orin vivo.

[0152] Viral Vectors:

[0153] Adenoviral Vectors

[0154] A viral gene delivery system useful in the present inventionutilizes adenovirus-derived vectors. Knowledge of the geneticorganization of adenovirus, a 36 kb, linear and double-stranded DNAvirus, allows substitution of a large piece of adenoviral DNA withforeign sequences up to 8 kb. In contrast to retrovirus, the infectionof adenoviral DNA into host cells does not result in chromosomalintegration because adenoviral DNA can replicate in an episomal mannerwithout potential genotoxicity. Also, adenoviruses are structurallystable, and no genome rearrangement has been detected after extensiveamplification. Adenovirus can infect virtually all epithelial cellsregardless of their cell cycle stage. So far, adenoviral infectionappears to be linked only to mild disease such as acute respiratorydisease in the human.

[0155] Adenovirus is particularly suitable for use as a gene transfervector because of its mid-sized genome, ease of manipulation, hightiter, wide target-cell range, and high infectivity. Both ends of theviral genome contain 100-200 base pair (bp) inverted terminal repeats(ITR), which are cis elements necessary for viral DNA replication andpackaging. The early (E) and late (L) regions of the genome containdifferent transcription domains that are divided by the onset of viralDNA replication. The E1 region (E1A and E1B) encodes proteinsresponsible for the regulation of transcription of the viral genome anda few cellular genes. The expression of the E2 region (E2A and E2B)results in the synthesis of the proteins for viral DNA replication.These proteins are involved in DNA replication, late gene expression,and host cell shut off (Renan (1990) Radiotherap. Oncol. 19:197). Theproducts of the late genes, including the majority of the viral capsidproteins, are expressed only after significant processing of a singleprimary transcript issued by the major late promoter (MLP). The MLP(located at 16.8 m.u.) is particularly efficient during the late phaseof infection, and all the mRNAs issued from this promoter possess a 5′tripartite leader (TL) sequence which makes them preferred mRNAs fortranslation.

[0156] The genome of an adenovirus can be manipulated such that itencodes a gene product of interest, but is inactivated in terms of itsability to replicate in a normal lytic viral life cycle (see, forexample, Berkner et al., (1988) BioTechniques 6:616; Rosenfeld et al.,(1991) Science 252:431-434; and Rosenfeld et al., (1992) Cell68:143-155). Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3,Ad7 etc.) are well known to those skilled in the art. Recombinantadenoviruses can be advantageous in certain circumstances in that theyare not capable of infecting nondividing cells and can be used to infecta wide variety of cell types, including airway epithelium (Rosenfeld etal., (1992) cited supra), endothelial cells (Lemarchand et al., (1992)PNAS USA 89:6482-6486), hepatocytes (Herz and Gerard, (1993) PNAS USA90:2812-2816) and muscle cells (Quantin et al., (1992) PNAS USA89:2581-2584). Adenovirus vectors have also been used in vaccinedevelopment (Grunhaus and Horwitz (1992) Seminar in Virology 3:237;Graham and Prevec (1992) Biotechnology 20:363). Experiments inadministering recombinant adenovirus to different tissues includetrachea instillation (Rosenfeld et al. (1991); Rosenfeld et al. (1992)Cell 68:143), muscle injection (Ragot et al. (1993) Nature 361:647),peripheral intravenous injection (Herz and Gerard (1993) Proc. Natl.Acad. Sci. U.S.A. 90:2812), and stereotactic inoculation into the brain(Le Gal La Salle et al. (1993) Science 254:988).

[0157] Furthermore, the virus particle is relatively stable and amenableto purification and concentration, and as above, can be modified so asto affect the spectrum of infectivity. Additionally, adenovirus is easyto grow and manipulate and exhibits broad host range in vitro and invivo. This group of viruses can be obtained in high titers, e.g.,10⁹-10¹¹ plaque-forming unit (PFU)/ml, and they are highly infective.The life cycle of adenovirus does not require integration into the hostcell genome. The foreign genes delivered by adenovirus vectors areepisomal, and therefore, have low genotoxicity to host cells. No sideeffects have been reported in studies of vaccination with wild-typeadenovirus (Couch et al., 1963; Top et al., 1971), demonstrating theirsafety and therapeutic potential as in vivo gene transfer vectors.Moreover, the carrying capacity of the adenoviral genome for foreign DNAis large (up to 8 kilobases) relative to other gene delivery vectors(Berkner et al., supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267).Most replication-defective adenoviral vectors currently in use andtherefore favored by the present invention are deleted for all or partsof the viral E¹ and E3 genes but retain as much as 80% of the adenoviralgenetic material (see, e.g., Jones et al., (1979) Cell 16:683; Berkneret al., supra; and Graham et al., in Methods in Molecular Biology, E. J.Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp. 109-127).Expression of the inserted gene can be under control of, for example,the E1A promoter, the major late promoter (MLP) and associated leadersequences, the viral E3 promoter, or exogenously added promotersequences.

[0158] Other than the requirement that the adenovirus vector bereplication defective, or at least conditionally defective, the natureof the adenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in themethod of the present invention. This is because Adenovirus type 5 is ahuman adenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector. As stated above, thetypical vector according to the present invention is replicationdefective and will not have an adenovirus E1 region. Thus, it will bemost convenient to introduce the nucleic acid of interest at theposition from which the E1 coding sequences have been removed. However,the position of insertion of the nucleic acid of interest in a regionwithin the adenovirus sequences is not critical to the presentinvention. For example, the nucleic acid of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors asdescribed previously by Karlsson et. al. (1986) or in the E4 regionwhere a helper cell line or helper virus complements the E4 defect.

[0159] A preferred helper cell line is 293 (ATCC Accession No. CRL1573).This helper cell line, also termed a “packaging cell line” was developedby Frank Graham (Graham et al. (1987) J. Gen. Virol. 36:59-72 and Graham(1977) J.General Virology 68:937-940) and provides E1A and E1B in trans.However, helper cell lines may also be derived from human cells such ashuman embryonic kidney cells, muscle cells, hematopoietic cells or otherhuman embryonic mesenchymal or epithelial cells. Alternatively, thehelper cells may be derived from the cells of other mammalian speciesthat are permissive for human adenovirus. Such cells include, e.g., Verocells or other monkey embryonic mesenchymal or epithelial cells.

[0160] Various adenovirus vectors have been shown to be of use in thetransfer of genes to mammals, including humans. Replication-deficientadenovirus vectors have been used to express marker proteins and CFTR inthe pulmonary epithelium. Because of their ability to efficiently infectdividing cells, their tropism for the lung, and the relative ease ofgeneration of high titer stocks, adenoviral vectors have been thesubject of much research in the last few years, and various vectors havebeen used to deliver genes to the lungs of human subjects (Zabner etal., Cell 75:207-216, 1993; Crystal, et al., Nat Genet. 8:42-51, 1994;Boucher, et al., Hum Gene Ther 5:615-639, 1994). The first generationE1a deleted adenovirus vectors have been improved upon with a secondgeneration that includes a temperature-sensitive E2a viral protein,designed to express less viral protein and thereby make the virallyinfected cell less of a target for the immune system (Goldman et al.,Human Gene Therapy 6:839-851,1995). More recently, a viral vectordeleted of all viral open reading frames has been reported (Fisher etal., Virology 217:11-22, 1996). Moreover, it has been shown thatexpression of viral IL-10 inhibits the immune response to adenoviralantigen (Qin et al., Human Gene Therapy 8:1365-1374, 1997).

[0161] Adenoviruses can also be cell type specific, i.e., infect onlyrestricted types of cells and/or express a transgene only in restrictedtypes of cells. For example, the viruses comprise a gene under thetranscriptional control of a transcription initiation regionspecifically regulated by target host cells, as described e.g., in U.S.Pat. No. 5,698,443, by Henderson and Schuur, issued Dec. 16, 1997. Thus,replication competent adenoviruses can be restricted to certain cellsby, e.g., inserting a cell specific response element to regulate asynthesis of a protein necessary for replication, e.g., E1A or E1B.

[0162] DNA sequences of a number of adenovirus types are available fromGenbank. For example, human adenovirus type 5 has GenBank AccessionNo.M73260. The adenovirus DNA sequences may be obtained from any of the42 human adenovirus types currently identified. Various adenovirusstrains are available from the American Type Culture Collection,Rockville, Md., or by request from a number of commercial and academicsources. A transgene as described herein may be incorporated into anyadenoviral vector and delivery protocol, by the same methods(restriction digest, linker ligation or filling in of ends, andligation) used to insert the CFTR or other genes into the vectors.

[0163] Adenovirus producer cell lines can include one or more of theadenoviral genes E1, E2a, and E4 DNA sequence, for packaging adenovirusvectors in which one or more of these genes have been mutated or deletedare described, e.g., in PCT/US95/15947 (WO 96/18418) by Kadan et al.;PCT/US95/07341 (WO 95/346671) by Kovesdi et al.; PCT/FR94/00624(WO94/28152) by Imler et al.; PCT/FR94/00851 (WO 95/02697) byPerrocaudet et al., PCT/US95/14793 (WO96/14061) by Wang et al.

[0164] AAV Vectors

[0165] Another viral vector system useful for delivery of DNA is theadeno-associated virus (AAV). Adeno-associated virus is a naturallyoccurring defective virus that requires another virus, such as anadenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review, see Muzyczka etal., Curr. Topics in Micro. and Immunol. (1992) 158:97-129).

[0166] AAV has not been associated with the cause of any disease. AAV isnot a transforming or oncogenic virus. AAV integration into chromosomesof human cell lines does not cause any significant alteration in thegrowth properties or morphological characteristics of the cells. Theseproperties of AAV also recommend it as a potentially useful human genetherapy vector.

[0167] AAV is also one of the few viruses that may integrate its DNAinto non-dividing cells, e.g., pulmonary epithelial cells or musclecells, and exhibits a high frequency of stable integration (see forexample Flotte et al., (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356;Samulski et al., (1989) J. Virol. 63:3822-3828; and McLaughlin et al.,(1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al., (1985) Mol. Cell. Biol. 5:3251-3260 can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al., (1984) PNAS USA 81:6466-6470; Tratschin et al., (1985)Mol. Cell. Biol. 4:2072-2081; Wondisford et al., (1988) Mol. Endocrinol.2:32-39; Tratschin et al., (1984) J. Virol. 51:611-619; and Flotte etal., (1993) J. Biol. Chem. 268:3781-3790).

[0168] The AAV-based expression vector to be used typically includes the145 nucleotide AAV inverted terminal repeats (ITRs) flanking arestriction site that can be used for subcloning of the transgene,either directly using the restriction site available, or by excision ofthe transgene with restriction enzymes followed by blunting of the ends,ligation of appropriate DNA linkers, restriction digestion, and ligationinto the site between the ITRs. The capacity of AAV vectors is about 4.4kb. The following proteins have been expressed using various AAV-basedvectors, and a variety of promoter/enhancers: neomycinphosphotransferase, chloramphenicol acetyl transferase, Fanconi's anemiagene, cystic fibrosis transmembrane conductance regulator, andgranulocyte macrophage colony-stimulating factor (Kotin, R. M., HumanGene Therapy 5:793-801, 1994, Table I). A transgene incorporating thevarious DNA constructs of this invention can similarly be included in anAAV-based vector. As an alternative to inclusion of a constitutivepromoter such as CMV to drive expression of the recombinant DNA encodingthe fusion protein(s), e.g. fusion proteins comprising an activationdomain or DNA-binding domain, an AAV promoter can be used (ITR itself orAAV p5 (Flotte, et al. J. Biol.Chem. 268:3781-3790, 1993)).

[0169] Such a vector can be packaged into AAV virions by reportedmethods. For example, a human cell line such as 293 can beco-transfected with the AAV-based expression vector and another plasmidcontaining open reading frames encoding AAV rep and cap (which areobligatory for replication and packaging of the recombinant viralconstruct) under the control of endogenous AAV promoters or aheterologous promoter. In the absence of helper virus, the rep proteinsRep68 and Rep78 prevent accumulation of the replicative form, but uponsuperinfection with adenovirus or herpes virus, these proteins permitreplication from the ITRs (present only in the construct containing thetransgene) and expression of the viral capsid proteins. This systemresults in packaging of the transgene DNA into AAV virions (Carter, B.J., Current Opinion in Biotechnology 3:533-539, 1992; Kotin, R. M, HumanGene Therapy 5:793-801, 1994)). Typically, three days aftertransfection, recombinant AAV is harvested from the cells along withadenovirus and the contaminating adenovirus is then inactivated by heattreatment.

[0170] Methods to improve the titer of AAV can also be used to expressthe transgene in an AAV virion. Such strategies include, but are notlimited to: stable expression of the ITR-flanked transgene in a cellline followed by transfection with a second plasmid to direct viralpackaging; use of a cell line that expresses AAV proteins inducibly,such as temperature-sensitive inducible expression or pharmacologicallyinducible expression. Alternatively, a cell can be transformed with afirst AAV vector including a 5′ ITR, a 3′ ITR flanking a heterologousgene, and a second AAV vector which includes an inducible origin ofreplication, e.g., SV40 origin of replication, which is capable of beinginduced by an agent, such as the SV40 T antigen and which includes DNAsequences encoding the AAV rep and cap proteins. Upon induction by anagent, the second AAV vector may replicate to a high copy number, andthereby increased numbers of infectious AAV particles may be generated(see, e.g, U.S. Pat. No. 5,693,531 by Chiorini et al., issued Dec. 2,1997. In yet another method for producing large amounts of recombinantAAV, a plasmid is used which incorporate the Epstein Barr NuclearAntigen (EBNA) gene, the latent origin of replication of Epstein Barrvirus (oriP) and an AAV genome. These plasmids are maintained as amulticopy extra-chromosomal elements in cells, such as in 293 cells.Upon addition of wild-type helper functions, these cells will producehigh amounts of recombinant AAV (U.S. Pat. No. 5,691,176 by Lebkowski etal., issued Nov. 25, 1997). In another system, an AAV packaging plasmidis provided that allows expression of the rep gene, wherein the p5promoter, which normally controls rep expression, is replaced with aheterologous promoter (U.S. Pat. No. 5,658,776, by Flotte et al., issuedAug. 19, 1997). Additionally, one may increase the efficiency of AAVtransduction by treating the cells with an agent that facilitates theconversion of the single stranded form to the double stranded form, asdescribed in Wilson et al., WO96/39530.

[0171] AAV stocks can be produced as described in Hermonat and Muzyczka(1984) PNAS 81:6466, modified by using the pAAV/Ad described by Samulskiet al. (1989) J. Virol. 63:3822. Concentration and purification of thevirus can be achieved by reported methods such as banding in cesiumchloride gradients, as was used for the initial report of AAV vectorexpression in vivo (Flotte, et al. J.Biol. Chem. 268:3781-3790, 1993) orchromatographic purification, as described in O'Riordan et al.,WO97/08298.

[0172] Methods for in vitro packaging AAV vectors are also available andhave the advantage that there is no size limitation of the DNA packagedinto the particles (see, U.S. Pat. No. 5,688,676, by Zhou et al., issuedNov. 18, 1997). This procedure involves the preparation of cell freepackaging extracts.

[0173] For additional detailed guidance on AAV technology which may beuseful in the practice of the subject invention, including methods andmaterials for the incorporation of a transgene, the propagation andpurification of the recombinant AAV vector containing the transgene, andits use in transfecting cells and mammals, see e.g. Carter et al, U.S.Pat. No. 4,797,368 (Jan. 10, 1989); Muzyczka et al, U.S. Pat. No.5,139,941 (Aug. 18, 1992); Lebkowski et al, U.S. Pat. No. 5,173,414(Dec. 22, 1992); Srivastava, U.S. Pat. No. 5,252,479 (Oct. 12, 1993);Lebkowski et al, U.S. Pat. No. 5,354,678 (Oct. 11, 1994); Shenk et al,U.S. Pat. No. 5,436,146(Jul. 25, 1995); Chatterjee et al, U.S. Pat. No.5,454,935 (Dec. 12, 1995), Carter et al WO 93/24641 (published Dec. 9,1993), and Natsoulis, U.S. Pat. No. 5,622,856 (Apr. 22, 1997). Furtherinformation regarding AAVs and the adenovirns or herpes helper functionsrequired can be found in the following articles. Berns and Bohensky(1987), “Adeno-Associated Viruses: An Update”, Advanced in VirusResearch, Academic Press, 33:243-306. The genome of AAV is described inLaughlin et al. (1983) “Cloning of infectious adeno-associated virusgenomes in bacterial plasmids”, Gene, 23: 65-73. Expression of AAV isdescribed in Beaton et al. (1989) “Expression from the Adeno-associatedvirus p5 and p19 promoters is negatively regulated in trans by the repprotein”, J. Virol., 63:4450-4454. Construction of rAAV is described ina number of publications: Tratschin et al. (1984) “Adeno-associatedvirus vector for high frequency integration, expression and rescue ofgenes in mammalian cells”, Mol. Cell. Biol., 4:2072-2081; Hermonat andMuzyczka (1984) “Use of adeno-associated virus as a mammalian DNAcloning vector: Transduction of neomycin resistance into mammaliantissue culture cells”, Proc. Natl. Acad. Sci. USA, 81:6466-6470;McLaughlin et al. (1988) “Adeno-associated virus general transductionvectors: Analysis of Proviral Structures”, J. Virol., 62:1963-1973; andSamulski et al. (1989) “Helper-free stocks of recombinantadeno-associated viruses: normal integration doqutre viral geneexpression”, J. Virol., 63:3822-3828. Cell lines that can be transformedby rAAV are those described in Lebkowski et al. (1988) “Adeno-associatedvirus: a vector system for efficient introduction and integration of DNAinto a variety of mammalian cell types”, Mol. Cell. Biol., 8:3988-3996.“Producer” or “packaging” cell lines used in manufacturing recombinantretroviruses are described in Dougherty et al. (1989) J. Virol.,63:3209-3212; and Markowitz et al. (1988) J. Virol., 62:1120-1124.

[0174] Hybrid Adenovirus-AAV Vectors

[0175] Hybrid Adenovirus-AAV vectors represented by an adenovirus capsidcontaining a nucleic acid comprising a portion of an adenovirus, and 5′and 3′ ITR sequences from an AAV which flank a selected transgene underthe control of a promoter. See e.g. Wilson et al, International PatentApplication Publication No. WO 96/13598. This hybrid vector ischaracterized by high titer transgene delivery to a host cell and theability to stably integrate the transgene into the host cell chromosomein the presence of the rep gene. This virus is capable of infectingvirtually all cell types (conferred by its adenovirus sequences) andstable long term transgene integration into the host cell genome(conferred by its AAV sequences).

[0176] The adenovirus nucleic acid sequences employed in the this vectorcan range from a minimum sequence amount, which requires the use of ahelper virus to produce the hybrid virus particle, to only selecteddeletions of adenovirus genes, which deleted gene products can besupplied in the hybrid viral process by a packaging cell. For example, ahybrid virus can comprise the 5′ and 3′ inverted terminal repeat (ITR)sequences of an adenovirus (which function as origins of replication).The left terminal sequence (5′) sequence of the Ad5 genome that can beused spans bp 1 to about 360 of the conventional adenovirus genome (alsoreferred to as map units 0-1) and includes the 5′ ITR and thepackaging/enhancer domain. The 3′ adenovirus sequences of the hybridvirus include the right terminal 3′ ITR sequence which is about 580nucleotides (about bp 35,353- end of the adenovirus, referred to asabout map units 98.4-100.

[0177] The AAV sequences useful in the hybrid vector are viral sequencesfrom which the rep and cap polypeptide encoding sequences are deletedand are usually the cis acting 5′ and 3′ ITR sequences. Thus, the AAVITR sequences are flanked by the selected adenovirus sequences and theAAV ITR sequences themselves flank a selected transgene. The preparationof the hybrid vector is further described in detail in published PCTapplication entitled “Hybrid Adenovirus-AAV Virus and Method of UseThereof”, WO 96/13598 by Wilson et al.

[0178] For additional detailed guidance on adenovirus and hybridadenovirus-AAV technology which may be useful in the practice of thesubject invention, including methods and materials for the incorporationof a transgene, the propagation and purification of recombinant viruscontaining the transgene, and its use in transfecting cells and mammals,see also Wilson et al, WO 94/28938, WO 96/13597 and WO 96/26285, andreferences cited therein.

[0179] Retroviruses

[0180] The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin (1990)Retroviridae and their Replication” In Fields, Knipe ed. Virology. NewYork: Raven Press). The resulting DNA then stably integrates intocellular chromosomes as a provirus and directs synthesis of viralproteins. The integration results in the retention of the viral genesequences in the recipient cell and its descendants. The retroviralgenome contains three genes, gag, pol, and env that code for capsidalproteins, polymerase enzyme, and envelope components, respectively. Asequence found upstream from the gag gene, termed psi, functions as asignal for packaging of the genome into virions. Two long terminalrepeat (LTR) sequences are present at the 5′ and 3′ ends of the viralgenome. These contain strong promoter and enhancer sequences and arealso required for integration in the host cell genome (Coffin (1990),supra).

[0181] In order to construct a retroviral vector, a nucleic acid ofinterest is inserted into the viral genome in the place of certain viralsequences to produce a virus that is replication-defective. In order toproduce virions, a packaging cell line containing the gag, pol, and envgenes but without the LTR and psi components is constructed (Mann et al.(1983) Cell 33:153). When a recombinant plasmid containing a human cDNA,together with the retroviral LTR and psi sequences is introduced intothis cell line (by calcium phosphate precipitation for example), the psisequence allows the RNA transcript of the recombinant plasmid to bepackaged into viral particles, which are then secreted into the culturemedia (Nicolas and Rubenstein (1988) “Retroviral Vectors”, In: Rodriguezand Denhardt ed. Vectors: A Survey of Molecular Cloning Vectors andtheir Uses. Stoneham:Butterworth; Temin, (1986) “Retrovirus Vectors forGene Transfer: Efficient Integration into and Expression of ExogenousDNA in Vertebrate Cell Genome”, In: Kucherlapati ed. Gene Transfer. NewYork: Plenum Press; Mann et al., 1983, supra). The media containing therecombinant retroviruses is then collected, optionally concentrated, andused for gene transfer. Retroviral vectors are able to infect a broadvariety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al. (1975) Virology67:242).

[0182] A major prerequisite for the use of retroviruses is to ensure thesafety of their use, particularly with regard to the possibility of thespread of wild-type virus in the cell population. The development ofspecialized cell lines (termed “packaging cells”) which produce onlyreplication-defective retroviruses has increased the utility ofretroviruses for gene therapy, and defective retroviruses are wellcharacterized for use in gene transfer for gene therapy purposes (for areview see Miller, A. D. (1990) Blood 76:271). Thus, recombinantretrovirus can be constructed in which part of the retroviral codingsequence (gag, pol, env) has been replaced by nucleic acid encoding afusion protein of the present invention, rendering the retrovirusreplication defective. The replication defective retrovirus is thenpackaged into virions which can be used to infect a target cell throughthe use of a helper virus by standard techniques. Protocols forproducing recombinant retroviruses and for infecting cells in vitro orin vivo with such viruses can be found in Current Protocols in MolecularBiology, Ausubel, F. M. et al., (eds.) Greene Publishing Associates,(1989), Sections 9.10-9.14 and other standard laboratory manuals.Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM whichare well known to those skilled in the art. A preferred retroviralvector is a pSR MSVtkNeo (Muller et al. (1991) Mol. Cell Biol. 11:1785and pSR MSV(XbaI) (Sawyers et al. (1995) J. Exp. Med. 181:307) andderivatives thereof. For example, the unique BamHI sites in both ofthese vectors can be removed by digesting the vectors with BamHI,filling in with Klenow and religating to produce pSMTN2 and pSMTX2,respectively, as described in PCT/US96/09948 by Clackson et al. Examplesof suitable packaging virus lines for preparing both ecotropic andamphotropic retroviral systems include Crip, Cre, 2 and Am.

[0183] Retroviruses have been used to introduce a variety of genes intomany different cell types, including neural cells, epithelial cells,endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrowcells, in vitro and/or in vivo (see for example Eglitis et al., (1985)Science 230:1395-1398; Danos and Mulligan, (1988) PNAS USA 85:6460-6464;Wilson et al., (1988) PNAS USA 85:3014-3018; Armentano et al., (1990)PNAS USA 87:6141-6145; Huber et al., (1991) PNAS USA 88:8039-8043; Ferryet al., (1991) PNAS USA 88:8377-8381; Chowdhury et al., (1991) Science254:1802-1805; van Beusechem et al., (1992) PNAS USA 89:7640-7644; Kayet al., (1992) Human Gene Therapy 3:641-647; Dai et al., (1992) PNAS USA89:10892-10895; Hwu et al., (1993) J. Immunol. 150:4104-4115; U.S. Pat.No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCTApplication WO 89/02468; PCT Application WO 89/05345; and PCTApplication WO 92/07573).

[0184] Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234,WO94/06920, and WO94/11524). For instance, strategies for themodification of the infection spectrum of retroviral vectors include:coupling antibodies specific for cell surface antigens to the viral envprotein (Roux et al., (1989) PNAS USA 86:9079-9083; Julan et al., (1992)J. Gen Virol 73:3251-3255; and Goud et al., (1983) Virology163:251-254); or coupling cell surface ligands to the viral env proteins(Neda et al., (1991) J. Biol. Chem. 266:14143-14146). Coupling can be inthe form of the chemical cross-linking with a protein or other variety(e.g. lactose to convert the env protein to an asialoglycoprotein), aswell as by generating fusion proteins (e.g. single-chain antibody/envfusion proteins). This technique, while useful to limit or otherwisedirect the infection to certain tissue types, and can also be used toconvert an ecotropic vector in to an amphotropic vector.

[0185] Other Viral Systems

[0186] Other viral vector systems that may have application in genetherapy have been derived from herpes virus, e.g., Herpes Simplex Virus(U.S. Pat. No. 5,631,236 by Woo et al., issued May 20, 1997), vacciniavirus (Ridgeway (1988) Ridgeway, “Mammalian expression vectors,” In:Rodriguez R L, Denhardt D T, ed. Vectors: A survey of molecular cloningvectors and their uses. Stoneham: Butterworth,; Baichwal and Sugden(1986) “Vectors for gene transfer derived from animal DNA viruses:Transient and stable expression of transferred genes,” In: KucherlapatiR, ed. Gene transfer. New York: Plenum Press; Coupar et al. (1988) Gene,68:1-10), and several RNA viruses. Preferred viruses include analphavirus, a poxvirus, an arena virus, a vaccinia virus, a polio virus,and the like. In particular, herpes virus vectors may provide a uniquestrategy for persistence of the recombinant gene in cells of the centralnervous system and ocular tissue (Pepose et al., (1994) InvestOphthalmol Vis Sci 35:2662-2666). They offer several attractive featuresfor various mammalian cells (Friedmann (1989) Science, 244:1275-1281Ridgeway, 1988, supra; Baichwal and Sugden, 1986, supra; Coupar et al.,1988; Horwich et al.(1990) J.Virol., 64:642-650).

[0187] With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990, supra).This suggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. recently introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas cotransfected with wild-type virus into an avian hepatoma cell line.Culture media containing high titers of the recombinant virus were usedto infect primary duckling hepatocytes. Stable CAT gene expression wasdetected for at least 24 days after transfection (Chang et al. (1991)Hepatology, 14:124A).

[0188] Administration of Viral Vectors

[0189] Generally the viral particles are transferred to a biologicallycompatible solution or pharmaceutically acceptable delivery vehicle,such as sterile saline, or other aqueous or non-aqueous isotonic sterileinjection solutions or suspensions, numerous examples of which are wellknown in the art, including Ringer's, phosphate buffered saline, orother similar vehicles. Delivery of the recombinant viral vector can becarried out via any of several routes of administration, includingintramuscular injection, intravenous administration, subcutaneousinjection, intrahepatic administration, catheterization (includingcardiac catheterization), intracranial injection,nebulization/inhalation or by instillation via bronchoscopy.

[0190] Preferably, the DNA or recombinant virus is administered insufficient amounts to transfect cells within the recipient's targetcells, including without limitation, muscle cells, liver cells, variousairway epithelial cells and smooth muscle cells, neurons, cardiac musclecells, etc. and provide sufficient levels of transgene expression toprovide for observable ligand-responsive secretion of a target protein,preferably at a level providing therapeutic benefit without undueadverse effects.

[0191] Optimal dosages of DNA or virus depends on a variety of factors,as discussed previously, and may thus vary somewhat from patient topatient. Again, therapeutically effective doses of viruses areconsidered to be in the range of about 20 to about 50 ml of salinesolution containing concentrations of from about 1×10⁷ to about 1×10¹⁰pfu of virus/ml, e.g. from 1×10⁸ to 1×10⁹ pfu of virus/ml.

[0192] Uses

[0193] In one application, cells engineered in accordance with theinvention are used to produce a target protein in vitro. In suchapplications, the cells are cultured or otherwise maintained untilproduction of the target protein is desired. At that time, theappropriate ligand is added to the culture medium, in an amountsufficient to cause the desired level of target protein production. Theprotein so produced may be recovered from the medium or from the cells,and may be purified from other components of the cells or medium asdesired.

[0194] Proteins for commercial and investigational purposes are oftenproduced using mammalian cell lines engineered to express the protein.The use of mammalian cells, rather than bacteria, insect or yeast cells,is indicated where the proper function of the protein requirespost-translational modifications not generally performed bynon-mammalian cells. Examples of proteins produced commercially this wayinclude, among others, erythropoietin, BMP-2, tissue plasminogenactivator, Factor VIII:c, Factor IX, and antibodies.

[0195] In other applications, cells within an animal host or humansubject are engineered in accordance with the invention, or cells soengineered are introduced into the animal or human subject, in eithercase, to prepare the recipient for ligand-mediated regulation ofsecretion of a therapeutic protein. In the case of non-human animals,this can be done as part of veterinary treatment of the animal or tocreate an animal model for a variety of research purposes. In the caseof human subjects, this can be done as part of a therapeutic orprophylactic treatment program.

[0196] This invention is applicable to a variety of treatmentapproaches. For example, the target protein, e.g. a therapeutic protein,to be regulated can be an endogenous protein or a heterologous protein,and its secretion may be activated by addition of ligand.

[0197] In some cases the target protein is a factor necessary for theproliferation and/or differentiation of one or more cell types ofinterest. For example, it may be desirable to stimulate the secretion ofgrowth factors and lymphokines in a subject in which at least some ofthe blood cells have been destroyed, e.g., by radiotherapy orchemotherapy. For example, secretion of erythropoietin stimulates theproduction of red blood cells, secretion of G-CSF stimulates theproduction of granulocytes, secretion of GM-CSF stimulates theproduction of various white blood cells, etc. Similarly in diseases orconditions in which one or more specific cell types are destroyed by thedisease process, e.g., in autoimmune diseases, the specific cells can bereplenished by stimulating secretion of one or more factors stimulatingproliferation of these cells. The method of the invention can also beused to increase the number of lymphocytes in a subject having AIDS,such as by stimulating secretion of lymphokines, e.g., IL-4, whichstimulates proliferation of certain T helper (Th) cells.

[0198] In other cases, the target protein is a hormone or endorphinwhich must be delivered rapidly and efficiently to its site of action.For example, patients with insulin-dependent diabetes mellitus (IDDM)must artificially maintain physiological levels of insulin in thebloodstream. It would be highly desirable to replace frequent insulininjections with a regulated expression system in which the patient couldrapidly produce his/her own insulin when needed. Current regulatedexpression systems rely on transcriptional mechanisms, in which proteinlevels increase about 12-16 hours after addition of ligand. In contrast,the present invention would allow delivery of insulin to the appropriatesite within 20-30 minutes after ligand binding. As in the case ofinsulin, the invention described herein could be used to treat anycondition which would benefit from rapid delivery of a therapeuticprotein. For example, this invention would be useful for delivery of anyprotein whose biology requires pulsatile or diurnal delivery. Suchproteins include, among others, parathyroid hormone or growth hormone.Other uses include delivery of proteins for inflammatory, flaring-typediseases, such as rheumatoid arthritis, inflammatory bowel disease, etc.Examples of such therapeutics would be antibodies to TNF, soluble TNFR,and IL-1RA. More generally, patients would benefit from regulatedsecretion of any “on-demand” or self-medicating scenario, like insulin(see above) or other agents for managing blood glucose; anti-painpeptides; inflammation (see above); leptin; contraception e.g.,antibodies to LHRH.

[0199] Methods for Identifying CRDs

[0200] Methods are disclosed below for the identification, validationand improvement of CRD candidates of each in each of the three classesdescribed earlier.

[0201] 1. CRDs Comprising Natural Examples of Proteins Retained inSecretory Compartments in a Small-molecule Reversible Manner

[0202] Candidate CRDs of this class include any naturally secretedprotein or subdomain thereof. Such proteins can typically be identifiedby the presence of a secretion signal sequence at the start of theircoding sequence. The characteristics of such signal sequences are wellknown and computational algorithms are available to assist in theiridentification. Using these methods, secreted proteins can be identifiedfrom searches of sequence databases. A preferred subset of secretedproteins are those that are known to bind small molecules, or arepredicted to do so by their homology to other small molecule-bindingproteins. The small molecule may be a ligand or substrate that istransiently bound to the protein during its normal function, or it maybe a cofactor that normally remains permanently bound. In either case,these small molecules provides a starting point for identifying ligandsfor the candidate CRD. In some cases (an example is rat RBP), smallmolecule-mediated release of the protein from secretory compartments mayalready be documented in the scientific literature.

[0203] To test whether a candidate protein can function as a CRD, DNAencoding the candidate polypeptide is amplified by PCR or RT-PCR usingstandard methods from an appropriate source, such as genomic DNA ortotal or poly A+RNA isolated from an appropriate cellular source, or acDNA or genomic DNA library. PCR primers are engineered to includerestriction sites allowing insertion into a vector for expression inmammalian cells, or other eukaryotic cells of interest. Alternativelythe sequence of interest can be isolated as a restriction fragment. ThePCR or restriction fragment is then cloned in frame into the polylinkerof an expression vector. A preferred vector is of the form shown in FIG.10A, where hCMV indicates the human CMV immediate early promoter andenhancer, SS indicates a signal sequence, poly is a polylinker region,FCS is a furin cleavage site, and hGH is a cDNA for human growthhormone. Components of this vector can be substituted as appropriate:for example, FCS can be replaced with alternative TGN protease cleavagesites, and hGH can be replaced with other secreted proteins that can beeasily detected and are therefore useful as reporter proteins, such assecreted alkaline phosphatase (SEAP) or erythropoietin (EPO).Optionally, an epitope tag allowing immunochemical detection of theprotein (for example the FLAG sequence: IBI/Kodak) can be included inthe vector sequence or incorporated via either PCR primer.

[0204] To determine whether the candidate polypeptide acts as a CRD, theexpression vector is introduced into cells in culture using standardtechniques, for example lipofection. After 24 hours, an aliquot ofculture medium is removed and assayed for presence of hGH using standardtechniques (Rivera et al., 1996). Then, new medium containing variousconcentrations of candidate CRD ligand are added. After a further periodof 2-24 hours, medium is again sampled for presence of hGH. CRD-likeactivity of the candidate polypeptide is indicated by a low level of hGHin the culture medium in the absence of compound, and increased amountsin the presence of compound. Suitable candidate CRD ligands toinvestigate include compounds that are known ligands of the proteinunder study (for example retinol for RBP), and chemically relatedmolecules that may have usefully different properties, such as cellpermeability or effects on ER retention of the protein (for examplediverse retinoids for RBP). Suitable concentrations of these ligands toinvestigate are in the range 1 pM to 1 mM.

[0205] An important approach for optimizing the effectiveness of CRDcandidates is the reiteration of those domains in multiple copies, toattempt to amplify any conditional retention effect. It is anticipated,for example, that some proteins will be ‘retarded’ in the secretorypathway in the absence of ligand, but not completely retained—that is,retention will be “leaky”. In some applications of the invention thiswill be desirable; in others, tightly repressed protein production inthe absence of drug will be needed. In these cases, reiterating the CRDmay augment the ability to cause retention of the heterologous protein.Thus, the experiments described above will optionally be repeated onconstructs that harbor different numbers of concatenated candidate CRDs:typically between one and eight.

[0206] Additional controls that can be performed to verify the activityof a CRD discovered through the above methods include immunochemicaldetection of the CRD and hGH domains inside cells treated or not treatedwith the CRD ligand, to confirm that the proteins are retained insidethe secretory apparatus. These experiments use standard cell fixingprocedures followed by immunofluorescence. Also, the secreted hGH can bechecked for correct processing from the fusion protein by size analysisusing SDS-PAGE followed by immunoblot with anti-hGH antibodies. For amore exact check, the hGH can be purified (eg. on an hGH binding proteinaffinity column), and then analyzed for molecular weight by massspectrometry and for correct processing by immobilization on PVDFfollowed by N-terminal sequence analysis.

[0207] Although the search for CRDs will typically focus on thoseproteins that are naturally secreted, and further on that subset ofsecreted proteins with known small molecule-binding activities, anypolypeptide can be tested using the methods described above. Thus aprotein that is not naturally secreted, but that has a known smallmolecule binding activity, can be cloned into the FCS-hGH expressionvector and tested for CRD behavior that can be reversed by that smallmolecule (or related molecules). Most generally, anypolypeptide—including one that is apparently not normally secreted, andthat has no known small molecule binding activity—can be tested. Inthese cases, the candidate CRD-FCS-hGH expression construct can be firsttested for retention of hGH. If retention is observed, cells containingthe construct can be challenged in separate experiments with a diverseset of candidate small molecules in order to identify a molecule thatcan promote secretion of the retained fusion proteins. Suitable sets ofmolecules include collections of natural products, and the members ofsynthetic or semi-synthetic combinatorial libraries. Screening may beexpedited by arraying cells in 96- or 384-well plates to enable robotichigh-throughput set-up and analysis of experiments.

[0208] 2. CRDs that are Mutants of a Natural Protein, Chosen for theProperty of being Selectively Retained in the Absence of a Given SmallMolecule

[0209] Screening methods for such CRDs follow on naturally from themethods described above. A polypeptide of interest is cloned into theFCS-hGH fusion expression vector described above. Again, preferredpolypeptides are those with known small molecule-binding activities.Individual mutants of the candidate CRD are engineered by standardmethods. These mutant constructs are then iteratively assayed for (i)the retention of hGH and (ii) the secretion of hGH upon addition of asmall molecule. Choice of small molecules to test, and theirconcentrations, are as described above. Assays on many mutants can beperformed simultaneously by using multi-well plate assays.

[0210] Mutations can be chosen to optimize the likelihood of inducing achange in the properties of the protein that results in conditionalretention. Mutations of particular interest are those anticipated todisrupt the efficient folding of the protein: such proteins might besubject to retention via the ER quality control system. Examplemutations include gain-of-size mutations of side chains that constitutethe hydrophobic core of the protein; and alterations of other residuesof critical importance in secondary or tertiary structural features,such as glycine residues at beta-turn motifs. Other amino acids ofinterest are those that form, or are close to, the small moleculebinding site. Mutants with reduced folding efficiency are preferredbecause such changes are most likely to be stabilized by binding of asmall molecule, providing a mechanism for selective smallmolecule-mediated release of retained proteins. Thus, knowledge of thethree-dimensional structure of a candidate CRD can be of great use infocusing mutagenesis to key positions.

[0211] Both singly and multiply mutated proteins can be engineered andtested. Often, the best variant protein will be altered at severalpositions. Identifying the best combination of changes at multipleresidues by iterative screening of mutants can be tedious andtime-consuming. An alternative is the use of selection procedures, inwhich a large set of mutants is created and then subjected en masse to aselection step to identify the best mutants directly. See Clackson andWells (Trends Biotech 1994 12: 173). To provide a means to directlyselect for proteins that act as CRDs, the expression vector describedabove is altered by exchanging the hGH coding sequence for DNA encodinga cell surface marker, such as CD2 or the p75 low affinity nerve growthfactor receptor. The extracellular and transmembrane domains of cellsurface marker are included, but most of the intracellular domain ispreferably deleted to remove the potential for signaling through thereceptor. A suitable expression vector using p75 is shown in FIG. 10B,where ECD and TM are respectively the extracellular and transmembranedomains of p75.

[0212] To select CRDs from a large set of candidates, genes encoding thecandidates are ligated into the polylinker to create a library. Thelibrary is introduced into mammalian cells by established methods.Methods should ideally be chosen that (i) lead to a low number ofvariants being introduced into each cell, so that the properties ofvariants can be tested individually, and (ii) provide stableintroduction of the vector so that cells can be propagated and selectedthrough multiple rounds. A preferred approach is therefore to constructthe library in a retroviral vector followed by retroviral infection ofcells, since this results single- or low-copy stable integration of thevector.

[0213] Selection of CRDs can be performed directly or indirectly. Directscreening is performed using a fluorescence-activated cell sorter, intwo stages. In the first stage, cells harboring the library of CRDcandidates are grown in culture and then incubated with afluorescently-labeled antibody to the p75 ECD. Cells containing a clonefor an active CRD will not bind, as p75 will be retained in thesecretory apparatus. However cells harboring ineffective CRDs will bindas the protein will not be retained. The labeled cells are sorted byFACS and cells that are not stained are gated and retrieved, and allowedto grow again in culture. The sort can optionally be repeated severaltimes with a progressively higher gate, in order to isolate the cellsexpressing lowest levels of p75. In the second stage, a candidate CRDligand (chosen as described above) is added and then the labelingprocess repeated. Now the cells with effective CRDs will be labeled,since the retained p75 will be released by the CRD ligand. The cells aresorted by FACS and the labeled cells are isolated. Again, the selectionstep can be repeated if desired. Once a suitable population of cells hasbeen isolated, the variants that are conferring the CRD activity can beidentified by isolating the genomic DNA of the cells followed by PCRamplification with primers located each side of the vector polylinker.The PCR products can then be cloned and sequenced. The ability of theidentified variants to act as CRDs can be confirmed by cloning themindividually into the hGH expression vector followed by testing asdescribed earlier. Indirect screening may be accomplished by determiningwhether the CRD directs surface localization of a membrane protein whichcan then activate a signaling pathway.

[0214] The mutants introduced can be targeted to the residues ofinterest indicated earlier, or can randomly incorporated. Severalsuitable methods for engineering sets of multiple mutants have beendescribed, including alanine-scanning mutagenesis (Cunningham and Wells(1989) Science 244 1081-1085), degenerate primer-mediated ‘Kunkel’mutagenesis (See eg. Lowman and Wells 1993 J Mol Biol 234: 563-578), PCRmisincorporation mutagenesis (see eg. Cadwell and Joyce (1992) PCR Meth.Applic. 2, 28-33), and DNA shuffling (Stemmer (1994) Nature 370389-391).

[0215] 3. CRDs that are Proteins that Self-aggregate in a SmallMolecule-reversible Manner.

[0216] Methods to identify proteins that interact with one another arewell known. A commonly used technique is the two-hybrid system, in whichone partner is fused to a DNA binding domain and the other to antranscriptional activation domain. Interaction of the partnersreconstitutes the transcription factor, activating transcription of areporter gene that can be identified by screening (eg. production ofbeta-galactosidase or SEAP) and/or that leads to cell survival andtherefore provides a means for selecting for interacting partners (eg.his gene transcription in a his- strain of yeast). Two-hybrid assays canbe performed in yeast or mammalian cells and methods are well known inthe art.

[0217] A preferred embodiment is based on the vectors and cellsdescribed by Rivera et al. (Nature Med 1996 2, 1028-1032). Twoexpression vectors are constructed for chimeric transcription factors inwhich the candidate CRD is fused to the hybrid DNA domain ZFHD1 (in onecase) and to an activiation domain of NF-kB p65 subunit, such as aminoacids 361-550 (in the other). These vectors are transiently or stablytransfected into mammalian cells, for example HT1080 cells, togetherwith a SEAP reporter gene under the control of ZFHD1 binding sites.Aggregation of the candidate CRDs results in reconstitution of an activetranscription factor and therefore prediction of SEAP. Once aself-aggregating protein has been identified in this way, addition ofcandidate CRD ligand can be used to examine whether the aggregates canbe dissociated with ligand. Reduction in the production of SEAP uponaddition of ligand would indicate this activity. Any polypeptide can bechosen for testing in this way for CRD activity, but preferred proteinsto try are those that already have known small molecule bindingactivity. In these cases the known binding ligands provide a startingpoint for choosing compounds that might disaggregate bound protein.

[0218] As before, an important additional configuration to explore isthe concatenation of candidate CRDs. Presence of more than oneaggregating domain may increase the apparent affinity of the aggregativeinteraction by virtue of the avidity effect.

[0219] Either natural or mutated proteins can be tested for CRDactivity. Mutants of natural proteins are likely to provide good sourcesof CRDs as examples are known on the literature of aggregative activityinduced by point mutations: for example sickle-cell hemoglobin, oralpha-1 antitrypsin as described earlier. Thus, large sets of mutants ofa candidate protein can be cloned into two-hybrid vectors as describedabove, and tested for aggregative activity that can be reduced byaddition of a small molecule. The criteria that dictate choice ofpositions to mutate will largely be the same as those described abovefor screening for CRDs directly in a secretion system (2 above); inaddition, mutants that aggregate might be provided by converting polarsurface residues to less polar amino ones. Single or multiple mutantscan be engineered, using methods as described above.

[0220] Selection schemes for CRDs can also be devised. In these cases,libraries of mutant proteins are cloned into two hybrid vectors andanalyzed en masse for CRD activity. These experiments are most easilyperformed in yeast and methods for two-hybrid selections are well knownin the art. For example, expression vectors for mutants of candidateCRDs, fused to GAL4 DNA binding domain or activation domain vectors, andtransformed into a his-deficient yeast reporter strain harboring a hisgene under the control of GAL4 binding sites. Plating the library onhis-deficient medium will result in growth only of cells that containinteracting CRDs on the two chimeric transcription factors. Thesepositives can then be replica plated onto plates containing increasingamounts of candidate CRD ligands, to identify those CRDs whoseinteractions can be disrupted by small molecules. Such proteins arecandidates for use as CRDs.

[0221] A complication with the above selection scheme is the desire tohave the same mutant fused to both the DNA binding and activationdomains, in order to identify proteins that self-aggregate. To achievethis, the expression vector for the chimeric proteins can be modified toallow a mutant gene to be joined to both transcription domains at thelevel of splicing. The domains of interest are encoded in separateexons. An outline of a suitable vector is shown in FIG. 10C. CRD and isthe candidate CRD: a library of candidates (eg mutant proteins) isinserted here. DBD and AD are the DNA binding and activation domains ofa transcription factor. A and D indicate donor and acceptor splicesites. stop indicates a translational stop codon. By equipping the DBDwith a suboptimal splice acceptor site, the CRD exon will be spliced toboth DBD and AD exons. Thus, in each cell fusion proteins will beexpressed in which the AD and DBD are both fused to an identical CRDcandidate.

[0222] An alternative format for selection of self-aggregating proteinsis the lambda repressor fusion system in E.coli (Hu et al. 1990 Science250:1400-1403; for review see Hu 1995 Structure 3: 431-433). Thisstrategy utilizes the fact that bacteriophage lambda repressor cI bindsto DNA as a homodimer and that binding of such homodimers to operatorDNA prevents transcription of phage genes involved in the lytic pathwayof the phage life cycle. Thus, bacterial cells expressing functionallambda repressor are immune to lysis by superinfecting lambdabacteriophage. Repressor protein comprises an amino terminal DNA bindingdomain (amino acids 1-92) joined by a 40 amino acid flexible linker to aC-terminal dimerization domain. The isolated N-terminal domain bindsvery weakly to DNA sue to inefficient dimer formation. High affinity DNAbinding can be restored by fusing the domain to a heterlogousdimerization domain, such as the GCN4 leucine zipper. A selection systemis therefore possible in which phage immunity is used as a selection forinteracting proteins.

[0223] For example, to select CRDs from a library of candidates, thecandidates are cloned in frame with the repressor N-terminus and thelibrary transformed into E.coli. Genes for proteins that aggregate areisolated from colonies that survive on plates containing high titers oflambda phage. These colonies can then be restreaked on to platescontaining both lambda phage and candidate CRD ligand. If the liganddissociates the aggregates, the E.coli will now no lolnger grow on theseplates. Lambda repressor selection has several advantages foridentifying CRDs, including the fact that the system is suitable forscreening homodimers, and the large library sizes that can be obtainedthrough the use of E.coli.

[0224] Another way to directly test whether a protein can act as a CRDin living cells is to fuse its coding sequence to green fluorescentprotein (GFP) or variants thereof. Cells expressing such a fusionprotein can then be examined directly by fluorescent microscopy toexamine whether the CRD candidate appears to cause aggregates of theGFP. Candidate CRD ligands can then be added to determine whether theaggregates then dissociate. Once a CRD candidate has been identified byany of these methods, it can be tested for activity as a CRD by use ofthe methods outlined in section 1.

[0225] Pharmaceutical Compositions & Their Administration to SubjectsContaining Engineered Cells

[0226] Administration

[0227] The ligand may be administered to a human or non-human subjectusing pharmaceutically acceptable materials and methods ofadministration. Various formulations, routes of administration, dose anddosing schedule may be used for the administration of ligand, dependingupon factors such as the condition and circumstances of the recipient,the response desired, the biological half-life and bioavailability ofthe ligand, the biological half-life and specific activity of the targetprotein product, the number and location of engineered cells present,etc. The drug may be administered parenterally, or more preferablyorally. For use in this invention, the most preferable route ofadministration are those in which a rapid onset of response occurs; suchmethods include, for example, sublingual, buccal, skin patch andinhalation. Dosage and frequency of administration will depend uponfactors such as described above. The drug may be taken orally as a pill,powder, or dispersion; buccally; sublingually; injected intravascularly,intraperitoneally, subcutaneously; or the like. The drug may beformulated using conventional methods and materials well known in theart for the various routes of administration. The precise dose andparticular method of administration will depend upon the above factorsand be determined by the attending physician or healthcare provider.

[0228] The particular dosage of the drug for any application may bedetermined in accordance with conventional approaches and procedures fortherapeutic dosage monitoring. A dose of the drug within a predeterminedrange is given and the patient's response is monitored so that the levelof therapeutic response and the relationship of protein secretion overtime may be determined. Depending on the expression levels observedduring the time period and the therapeutic response, one may adjust thelevel of subsequent dosing to alter the resultant expression level overtime or to otherwise improve the therapeutic response. This process maybe iteratively repeated until the dosage is optimized for therapeuticresponse. Where the drug is to be administered chronically, once amaintenance dosage of the drug has been determined, one may conductperiodic follow-up monitoring to assure that the overall therapeuticresponse continues to be achieved.

[0229] In the event that the activation by the drug is to be reversed,administration of drug may be suspended so that cells return to a basalrate of secretion. To effect a more active reversal of therapy, anantagonist of the drug may be administered. An antagonist is a compoundwhich binds to the drug or drug-binding domain to inhibit interaction ofthe drug with the fusion protein(s) and thus inhibit the downstreambiological event. Thus, in the case of an adverse reaction or the desireto terminate the therapeutic effect, an antagonist can be administeredin any convenient way, particularly intravascularly or byinhalation/nebulization, if a rapid reversal is desired.

[0230] Compositions

[0231] Drugs (i.e., the ligands) for use in this invention can exist infree form or, where appropriate, in salt form. The preparation of a widevariety of pharmaceutically acceptable salts is well-known to those ofskill in the art. Pharmaceutically acceptable salts of various compoundsinclude the conventional non-toxic salts or the quaternary ammoniumsalts of such compounds which are formed, for example, from inorganic ororganic acids of bases. The drugs may form hydrates or solvates. It isknown to those of skill in the art that charged compounds form hydratedspecies when lyophilized with water, or form solvated species whenconcentrated in a solution with an appropriate organic solvent.

[0232] The drugs can also be administered as pharmaceutical compositionscomprising a therapeutically (or prophylactically) effective amount ofthe drug, and a pharmaceutically acceptable carrier or excipient.Carriers include e.g. saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof, and are discussed ingreater detail below. The composition, if desired, can also containminor amounts of wetting or emulsifying agents, or pH buffering agents.The composition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation, or powder. The compositioncan be formulated as a suppository, with traditional binders andcarriers such as triglycerides. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc. Formulation may involve mixing, granulating and compressing ordissolving the ingredients as appropriate to the desired preparation.The pharmaceutical carrier employed may be, for example, either a solidor liquid.

[0233] Illustrative solid carriers include lactose, terra alba, sucrose,talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acidand the like. A solid carrier can include one or more substances whichmay also act as flavoring agents, lubricants, solubilizers, suspendingagents, fillers, glidants, compression aids, binders ortablet-disintegrating agents; it can also be an encapsulating material.In powders, the carrier is a finely divided solid which is in admixturewith the finely divided active ingredient. In tablets, the activeingredient is mixed with a carrier having the necessary compressionproperties in suitable proportions and compacted in the shape and sizedesired. The powders and tablets preferably contain up to 99% of theactive ingredient. Suitable solid carriers include, for example, calciumphosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch,gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose,polyvinylpyrrolidine, low melting waxes and ion exchange resins.

[0234] Illustrative liquid carriers include syrup, peanut oil, oliveoil, water, etc. Liquid carriers are used in preparing solutions,suspensions, emulsions, syrups, elixirs and pressurized compositions.The active ingredient can be dissolved or suspended in apharmaceutically acceptable liquid carrier such as water, an organicsolvent, a mixture of both or pharmaceutically acceptable oils or fats.The liquid carrier can contain other suitable pharmaceutical additivessuch as solubilizers, emulsifiers, buffers, preservatives, sweeteners,flavoring agents, suspending agents, thickening agents, colors,viscosity regulators, stabilizers or osmo-regulators. Suitable examplesof liquid carriers for oral and parenteral administration include water(partially containing additives as above, e.g. cellulose derivatives,preferably sodium carboxymethyl cellulose solution), alcohols (includingmonohydric alcohols and polyhydric alcohols, e.g. glycols) and theirderivatives, and oils (e.g. fractionated coconut oil and arachis oil).For parenteral administration, the carrier can also be an oily estersuch as ethyl oleate and isopropyl myristate. Sterile liquid carders areuseful in sterile liquid form compositions for parenteraladministration. The liquid carrier for pressurized compositions can behalogenated hydrocarbon or other pharmaceutically acceptable propellant.Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by, for example, intramuscular,intraperitoneal or subcutaneous injection. Sterile solutions can also beadministered intravenously. The drugs can also be administered orallyeither in liquid or solid composition form.

[0235] The carrier or excipient may include time delay material wellknown to the art, such as glyceryl monostearate or glyceryl distearatealong or with a wax, ethylcellulose, hydroxypropylmethylcellulose,methylmethacrylate and the like. When formulated for oraladministration, 0.01% Tween 80 in PHOSAL PG-50 (phospholipid concentratewith 1,2-propylene glycol, A. Nattermann & Cie. GmbH) may be used as anoral formulation for a variety of drugs for use in the practice of thisinvention.

[0236] A wide variety of pharmaceutical forms can be employed. If asolid carrier is used, the preparation can be tableted, placed in a hardgelatin capsule in powder or pellet form or in the form of a troche orlozenge. The amount of solid carrier will vary widely but preferablywill be from about 25 mg to about 1 g. If a liquid carrier is used, thepreparation will be in the form of a syrup, emulsion, soft gelatincapsule, sterile injectable solution or suspension in an ampule or vialor nonaqueous liquid suspension.

[0237] To obtain a stable water soluble dosage form, a pharmaceuticallyacceptable salt of the drug may be dissolved in an aqueous solution ofan organic or inorganic acid, such as a 0.3M solution of succinic acidor citric acid. Alternatively, acidic derivatives can be dissolved insuitable basic solutions. If a soluble salt form is not available, thecompound is dissolved in a suitable cosolvent or combinations thereof.Examples of such suitable dissolved in a suitable cosolvent orcombinations thereof. Examples of such suitable cosolvents include, butare not limited to, alcohol, propylene glycol, polyethylene glycol 300,polysorbate 80, glycerin, polyoxyethylated fatty acids, fatty alcoholsor glycerin hydroxy fatty acids esters and the like in concentrationsranging from 0-60% of the total volume.

[0238] Various delivery systems are known and can be used to administerthe drugs, or the various formulations thereof, including tablets,capsules, injectable solutions, encapsulation in liposomes,microparticles, microcapsules, etc. Preferred routes of administrationto a patient are oral, sublingual, transdermal (patch), intranasal,pulmonary or bucal. Methods of introduction also could include but arenot limited to dermal, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, epidural, ocular and (as is usuallypreferred) oral routes. The drug may be administered by any convenientor otherwise appropriate route, for example by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local. For ex vivo applications, thedrug will be delivered as a liquid solution to the cellular composition.

[0239] In a specific embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic to ease pain at theside of the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as alyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

[0240] In addition, in certain instances, it is expected that thecompound may be disposed within devices placed upon, in, or under theskin. Such devices include patches, implants, and injections whichrelease the compound into the skin, by either passive or active releasemechanisms.

[0241] Materials and methods for producing the various formulations arewell known in the art and may be adapted for practicing the subjectinvention. See e.g. U.S. Pat. Nos. 5,182,293 and 4,837,311 (tablets,capsules and other oral formulations as well as intravenousformulations) and European Patent Application Publication Nos. 0 649 659(published Apr. 26, 1995; rapamycin formulation for IV administration)and 0 648 494 (published Apr. 19, 1995; rapamycin formulation for oraladministration).

[0242] The effective dose of the drug will typically be in the range ofabout 0.01 to about 50 mg/kgs, preferably about 0.1 to about 10 mg/kg ofmammalian body weight, administered in single or multiple doses.Generally, the compound may be administered to patients in need of suchtreatment in a daily dose range of about 1 to about 2000 mg per patient.In embodiments in which the compound is rapamycin or an analog thereofwith some residual immunosuppressive effects, it is preferred that thedose administered be below that associated with undue immunosuppressiveeffects.

[0243] The amount of a given drug which will be effective in thetreatment or prevention of a particular disorder or condition willdepend in part on the severity of the disorder or condition, and can bedetermined by standard clinical techniques. In addition, in vitro or invivo assays may optionally be employed to help identify optimal dosageranges. Effective doses may be extrapolated from dose-response curvesderived from in vitro or animal model test systems. The precise dosagelevel should be determined by the attending physician or other healthcare provider and will depend upon well known factors, including routeof administration, and the age, body weight, sex and general health ofthe individual; the nature, severity and clinical stage of the disease;the use (or not) of concomitant therapies; and the nature and extent ofgenetic engineering of cells in the patient.

[0244] The drugs can also be provided in a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions. Optionally associatedwith such container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceutical or biological products, which notice reflects approval bythe agency of manufacture, use or sale for human administration.

[0245] The full contents of all references cited in this document,including references from the scientific literature, issued patents andpublished patent applications, are hereby expressly incorporated byreference.

[0246] The following examples contain important additional information,exemplification and guidance which can be adapted to the practice ofthis invention in its various embodiments and the equivalents thereof.The examples are offered by way of illustration only and should not beconstrued as limiting in any way. As noted throughout this document, theinvention is broadly applicable and permits a wide range of designchoices by the practitioner.

[0247] The practice of this invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,immunology, virology, pharmacology, chemistry, and pharmaceuticalformulation and administration which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

EXAMPLES Example 1

[0248] Generation of Domains and Vectors Used for Expression of F(36M)Fusion Proteins.

[0249] A. Expression Vectors:

[0250] Vectors for driving expression of fusion proteins were derivedfrom the mammalian expression vector pCGNN (Attar and Gilman, MCB12:2432-2443, 1992). Inserts cloned as XbaI-BamHI fragments into pCGNNare transcribed under the control of the human CMV promoter and enhancersequences (nucleotides −522 to +72 relative to the cap site), and areexpressed with an N-terminal nuclear localization sequence (NLS; fromSV40 T antigen) and epitope tag (a 16 amino acid portion of the H.influenzae hemaglutinin gene).

[0251] pCGNN was modified by site directed mutagenesis witholigonucleotides VR65, VR119, and VR120 to create pC4EN. The resultingplasmid has unique restriction sites upstream of the CMVenhancer/promoter region (MluI) and between the promoter and proteincoding region (EcoRI).

[0252] VR65: TCCCGCACCTCTTCGGCCAGCGaaTTccAGAAGCGCGTAT (SEQ ID NO.2)

[0253] VR119: GACTCACTATAGGaCGcgTTCGAGCTCGCCCC (SEQ ID NO. 3)

[0254] VR120: CATCATTTTGGCAAAGgATTCACTCCTCAGG (SEQ ID NO. 4)

[0255] Individual components of fusion proteins were generally producedas fragments containing an XbaI site immediately upstream of the firstcodon and a SpeI site, an in-frame stop codon, and a BamHI siteimmediately downstream of the last codon. Chimeric proteins comprisingmultiple components were assembled by stepwise insertion of XbaI-BamHIfragments into SpeI-BamHI-opened vectors or by insertion of XbaI-SpeIfragments into XbaI or SpeI-opened vectors.

[0256] B. F(36M) Domain

[0257] F(36M), in which the phenylalanine at amino acid 36 was changedto methionine, was created by mutagenizing a single FKBP domain, clonedinto pCGNN with upstream XbaI and downstream SpeI and BamHI sites(Rivera et al., Nat. Med 2:1028-1032, 1996) with oligo VR1 to createpCGNN-F(36M). Two, 3, 4 and 6 tandem copies of F(36M) were created bythe stepwise insertion of XbaI-BamHI fragments into SpeI-BamHI-openedvectors.

[0258] VR1: GATGGAAAGAAAatgGATTCCTCCCGG (SEQ ID NO. 5)

[0259] C. F(36M) Fusion Proteins: (FIG. 3)

[0260] (a) EGFP Fusions

[0261] EGFP coding sequence was amplified from pEGFP-1 (Clontech) witholigos VR2 and VR3. The resulting fragment, with upstream XbaI anddownstream SpeI sites was inserted into pCGN, a derivative of pCGNN thatlacks the SV40 nuclear localization sequence, to create pCGN-EGFP.

[0262] VR2: tctagaGTGAGCAAGGGCGAGGAG (SEQ ID NO. 6)

[0263] VR3: ggatccttaTTAACTAGTCTTGTACAGCTCGTCCATG (SEQ ID NO. 7)

[0264] F(36M)-EGFP fusions were created by inserting XbaI-SpeI fragmentscontaining 3, 4 or 6 copies of F(36M) into the XbaI site of pCGN-EGFP tocreate pCGN-F(36M)3-EGFP, pCGN-F(36M)4-EGFP, and pCGN-F(36M)6-EGFP.

[0265] (b) hGH Fusions

[0266] An hGH cDNA (506-81) was obtained by RT-PCR amplification of RNAexpressed from a cell line containing a genomic hGH gene (Rivera et al.,Nat. Med 2:1028-1032, 1996) using oligos VR109 and VR110 to amplify theregion from 40 bp upstream of the ATG to 60 bp after the stop codon. Theresulting HindIII to EcoRI fragment was cloned into Z12I-PL-2, aderivative of ZHWTx12-IL2-SEAP (Rivera et al., Nat. Med 2:1028-1032,1996) in which the SEAP gene and SV40 early intron and polyadenylationsignal were replaced by a polylinker and the SV40 late polyadenylationsignal.

[0267] VR109: aagcttACCACTCAGGGTCCTGTGG (SEQ ID NO. 8)

[0268] VR110: gaattcGTGGCAACTTCCA (SEQ ID NO. 9)

[0269] To construct hGH fusion proteins, Z12I-hGH-2 was mutagenized witholigos VR185, VR186, and VR187 to create i) an EcoRI site 32 bp upstreamof the ATG, ii) an XbaI site immediately after the last amino acid ofthe signal sequence and iii) a Spe I site immediately after the lastamino acid of hGH.

[0270] VR185: cacaggaccctGAATTCtaagcttgtggc (SEQ ID NO. 10)

[0271] VR186: ATAAGGGAATGGTtctagaGGCACTGCCCT (SEQ ID NO. 11)

[0272] VR187: atgccacccgggactagtGAAGCCACAGCTG (SEQ ID NO. 12)

[0273] Cloning the resulting EcoRI-SpeI fragment into pC4EN producedpC4S1-hGH which expresses hGH from the CMV enhancer. The XbaI-BamHIfragment of pC4S1-hGH was then replaced by XbaI-SpeI fragmentscontaining 2, 3, 4, or 6 copies of F(36M) and a SpeI-BamHI fragmentencoding the furin cleavage site-hGH fusion to generatepC4S1-F(36M)-FCS-hGH fusions.

[0274] A SpeI-BamHI fragment encoding an FCS-hGH fusion protein wasgenerated by amplification of the hGH cDNA with oligos VR4 and VR5.

[0275] VR4;actagtGCTAGAAACCGTCAGAAGAGATTCCCAACCATTCCCTTAAGC (SEQ ID NO.13)

[0276] VR5: ggatcccgggCTAGAAGCCACAGCTGCCCTC (SEQ ID NO. 14)

[0277] An XbaI-BamHI fragment containing the neo resistance genedownstream of the encephalomyocarditis virus internal ribosome entrysequence (IRES/Neo; Amara et al PNAS 94:10618-23, 1997) was insertedinto appropriate SpeI-BamHI-opened vectors to generatepC4S1-F(36M)-FCS-hGH/neo and pC4S1- hGH/neo vectors.

[0278] (c) Insulin Fusions

[0279] A human insulin cDNA was obtained by RT-PCR amplification ofhuman pancreas polyA+RNA (Clontech) using oligos VR220 and VR221 toamplify the region from 9 bp upstream of ATG (EcoRI) to 13 bp after stopcodon (BamHI). The resulting EcoRI-BamHI fragment was cloned into pC4ENto generate pC4-hIn.

[0280] VR220: cGAATTCttctgccATGGCCCTGTGGATGCGC (SEQ ID NO. 15)

[0281] VR221: cGGATCCgcaggctgcgtCTAGTTGCAGTAG (SEQ ID NO. 16)

[0282] A SpeI-BamHI fragment encoding an furin cleavage sequence-insulinfusion protein was generated by RT-PCR amplification with oligos VR222and VR221.

[0283] VR222: cACTAGTGCTAGAAACCGTCAGAAGAGATTTGTGAACCAACACCTGTGCGGC (SEQID NO. 17)

[0284] VR221: cGGATCCgcaggctgcgtCTAGTTGCAGTAG (SEQ ID NO. 18)

[0285] The wild type insulin gene and FCS-insulin fusion weremutagenized to i) alter amino acid B10 to Asp, ii) create a FCS at theB-C junction, and iii) create a FCS at the C-A junction, using oligosVR223, VR224, VR225, respectively.

[0286] VR223: CCTGTGCGGCTCAgACCTGGTGGAAGC (SEQ ID NO. 19)

[0287] VR224: CTTCTACACACCCAgGACCaagCGGGAGGCAGAGG (SEQ ID NO. 20)

[0288] VR225: CCCTGGAGGGGTCCCgGCAGAAGCGTGGC (SEQ ID NO. 21)

[0289] Mutation of pC4-hIn produced pC4-hIn-m3. The mutated FCS-insulinfusions were used to replace the FCS-hGH portion of thepC4S1-F(36M)-FCS-hGH fusions to create pC4S1-F(36M)-FCS-hIn-m3 fusions.

[0290] (d) LNGFR Fusions

[0291] EcoRI-SpeI fragments containing amino acids 1-274 of the humanlow affinity nerve growth factor receptor (LNGFR; Clackson et al., PNAS95:10437-42, 1998) and SpeI-BamHI fragments containing 3, 4, or 6 copiesof F(36M) were cloned into pC4EN to generate pC4LNGFR-F(36M) fusions.

[0292] (c) Transcription Factor Fusions

[0293] pCGNN-ZFHD1-F(36M) and pCGNN-F(36M)-p65 fusion proteins weregenerated as described for wild type FKBP fusions (Amara et al PNAS94:10618-23, 1997).

[0294] An XbaI-SpeI fragment containing 6 copies of F(36M) was insertedinto the XbaI or SpeI site of

[0295] pCGNN-ZFHD1-p65 to generate pCGNN- F(36M)6-ZFHD1-p65 and

[0296] pCGNN-ZFHD1-p65-F(36M)6.

[0297] pCGNNZFHD1

[0298] An expression vector for directing the expression of ZFHD1 codingsequence in mammalian cells was prepared as follows. Zif268 sequenceswere amplified from a cDNA clone by PCR using primers 5′Xba/Zif and3′Zif+G. Oct1 homeodomain sequences were amplified from a cDNA clone byPCR using primers 5′Not Oct HD and Spe/Bam 3′Oct. The Zif268 PCRfragment was cut with XbaI and NotI. The OctI PCR fragment was cut withNotI and BamHI. Both fragments were ligated in a 3-way ligation betweenthe XbaI and BamHI sites of pCGNN (Attar and Gilman, 1992) to makepCGNNZFHD1 in which the cDNA insert is under the transcriptional controlof human CMV promoter and enhancer sequences and is linked to thenuclear localization sequence from SV40 T antigen. The plasmid pCGNNalso contains a gene for ampicillin resistance which can serve as aselectable marker.

[0299] pCGNNZFHD1-p65

[0300] An expression vector for directing the expression in mammaliancells of a chimeric transcription factor containing the compositeDNA-binding domain, ZFHD1, and a transcription activation domain fromp65 (human) was prepared as follows. The sequence encoding theC-terminal region of p65 containing the activation domain (amino acidresidues 450-550) was amplified from pCGN-p65 using primers p65 5′ Xbaand p65 3′ Spe/Bam. The PCR fragment was digested with XbaI and BamHIand ligated between the the SpeI and BamH1 sites of pCGNN ZFHD1 to formpCGNN ZFHD-p65AD.

[0301] The P65 transcription activation sequence contains the followinglinear sequence: (SEQ ID NO. 22)CTGGGGGCCTTGCTTGGCAACAGCACAGACCCAGCTGTGTTCACAGACCTGGCATCCGTCGACAACTCCGAGTTTCAGCAGCTGCTGAACCAGGGCATACCTGTGGCCCCCCACACAACTGAGCCCATGCTGATGGAGTACCCTGAGGCTATAACTCGCCTAGTGACAGGGGCCCAGAGGCCCCCCGACCCAGCTCCTGCTCCACTGGGGGCCCCGGGGCTCCCCAATGGCCTCCTTTCAGGAGATGAAGACTTCTCCTCCATTGCGGACATGGACTTCTCAGCCCTGCTGAGTCAGATC AGCTCC

Example 2

[0302] Identification and Synthesis of a Ligand for the ConditionalRetention Domain F36M FKBP.

[0303] AP21998 and AP22542 are ligands of FKBP that have particularutility for CAD applications, because they bind with high affinity toF36M-FKBP but poorly to the wild-type protein, and are thus anticipatedto lead to minimal interactions with the endogenous proteins during invivo applications. The design and assay of such “bumped” ligands thattarget a hole created by truncating FKBP residue Phe36 have beendescribed (Clackson et al., Proc. Natl. Acad. Sci. USA 95:10437-10442,1998).

[0304] AP 21998 was prepared via DCC/DMAP-mediated coupling of thepreviously described acid AP 1867 (compound 5S in Clackson et al., Proc.Natl. Acad. Sci. USA 95:10437-10442, 1998) with commercially availableN,N-dimethyl-1,3-propanediamine (Scheme 1). AP 22542 was alsosynthesized by a DCC/DMAP-mediated coupling of acid AP 17362 withalcohol 3 (Scheme 2). Carbinol 3 itself was prepared via a three stepsequence as outlined in Scheme 2. The Claisen-Schmidt condensation of3,4-dimethoxybenzaldehyde and 3-acetylpyridine provided unsaturatedketone 1 as a crystalline solid in 68% yield. Transfer hydrogenation of1 utilizing ammonium formate as a hydrogen source provided the propanoneadduct 2 as a crystalline solid in 50% isolated yield. Finally, theenantioselective reduction of the aryl ketone moiety of 2 to the desiredR-configured carbinol 3 was achieved in 86% by reduction of 2 with(+)-b-chlorodiisopinocamphenylborane (DIP-Chloride™) (Chandrasekharan etal. J. Org. Chem. 50:5446, 1985). The synthesis of the acid component,AP 17362, was prepared as described in Scheme 3. The commerciallyavailable 3,4,5-trimethoxyphenylacetic acid was converted to the racemic2-arylbutane derivative 4 in 83% yield by alkylation with iodoethane ofthe NaHMDS-generated dianion of 3,4,5-trimethoxyphenylacetic acid in THFat 0 oC. Resolution of the acid by repetitive crystallization of the(−)-cinchonidine salt afforded optically enhanced 4S in 24% yield (48%theoretical) and of 91% ee. This resolved acid was then coupled withmethyl-L-pipecolate hydrochloride by use of 2-chloro-1-methylpyridiniumiodide (Mukaiyama's Reagent). The resulting coupled product was notisolated, but subjected to hydrolysis to afford the desired crystallineacid, AP 17362, in 42% overall yield and >99% de. X-ray structuralanalysis confirmed the absolute stereochemistry of the resolved2-arylbutane center as the S configuration.

[0305] AP 21998

[0306] A solution of AP 1867 (5.0 g, 7.21 mmol) in CH2Cl2 (5.0 mL) at 0°C. was treated with DCC (178 mg, 0.79 mmol) followed 30 min later byN,N-dimethyl-1,3-propanediamine (880 mg, 8.65 mmol) and DMAP (5 mg). Thereaction 5 mixture was allowed to warm to room temperature and stir for5 h, after which time the reaction mixture was diluted with EtOAc (50mL), filtered, and the filtrate extracted with a 5% aqueous citric acidsolution (3×20 ml). The acid extract was then made basic by the additionof solid NaHCO3 and extracted with EtOAc (3×50 mL). The organic extractwas dried over Na2SO4, filtered, and evaporated to afford a crudematerial which was flash chromatographed on silica gel (5% then 15%MeOH/CH2Cl2) to afford product (2.2 g, 39%) as a colorless foam:

[0307] IR (neat) 2940, 1735, 1650, 1510, 1460, 1240, 1130 cm⁻¹; ¹H NMR(CDCl3, 300 MHz) 7.78 (br t, J=5.1 Hz, 1 H), 7.19 (t, J=8.6 Hz, 1 H),6.92-6.65 (m, 6 H), 6.42 (s, 2 H), 5.63 (dd, J=8.0, 5.5 Hz, 1 H), 5.45(d, J=4.1 Hz, 1 H), 4.49 (s, 2 H), 3.86-3.70 (m, 16 H), 3.60 (t, J=7.0Hz, 1 H), 3.47-3.41 (m, 2 H), 2.82 (td, J=13.2, 2.4 Hz, 1 H), 2.62-2.29(m, 12 H), 2.16-1.23 (m, 10 H), 0.90 (t, J=7.3 Hz, 3 H); ¹³C NMR (CDCl3,75 MHz) 172.7, 170.6, 168.5, 157.5, 153.2, 148.9, 147.4, 142.3, 136.7,135.3, 133.4, 129.8, 120.2, 119.6, 113.9, 112.8, 111.8, 111.4, 105.1,75.7, 67.3, 60.8, 56.3, 56.0, 52.1, 50.7, 44.3, 43.5, 38.3, 37.4, 31.3,28.3, 26.8, 25.5, 25.4, 20.9, 12.5; LRMS (ES+): (M+H)⁺ 778; HRMS (FAB):(M+H)⁺ calcd: 778.4278, meas: 778.4299.

[0308] (E)-3-(3,4-Dimethoxyphenyl)-1-pyridin-3-yl-propenone (1): Asolution of 3,4-dimethoxybenzaldehyde (53.7 g, 323 mmol) and3-acetylpyridine (39.1 g, 323 mmol) in EtOH (400 mL) was treated withpiperdine (4.75 mL, 48 mmol) and heated at reflux for 4 days. Thereaction was then evaporated to a slurry and treated with water (400mL). The resulting solids were filtered, air dried, and recrystallizedfrom EtOAc/hexane to afford product (59.2 g, 68%) as a yellow coloredsolid: mp 111-112.5° C.; TLC (EtOAc) Rf=0.30; 1H NMR (CDCl3, 300 MHz)9.23 (d, J=1.8 Hz, 1 H), 8.79 (dd, J=4.8, 1.7 Hz, 1 H), 8.28 (dt, J=7.9,1.9 Hz, 1 H 7.79 (d, J=15.6 Hz, 1 H), 7.46-7.42 (m, 1 H), 7.35 (d,J=15.6 Hz, 1 H), 7.24 (dd, J=8.3, 1.9 Hz, 1 H), 7.68 (d, J=1.9 Hz, 1 H),6.91 (d, J=8.3 Hz, 1 H), 3.95 (s, 3 H), 3.93 (s, 3 H); 13C NMR (CDCl3,75 MHz) 189.0, 152.9, 151.9, 149.7, 149.4, 146.1, 135.8, 133.8, 127.5,123.6, 119.4, 111.2, 110.2, 56.0; LRMS (ES+) (M+H)+ 270; Anal. Calcd forC16H15NO3: C, 71.36; H, 5.61; N, 5.20. Found: C, 71.13; H, 5.70; N,4.95.

[0309] 3-(3,4-Dimethoxyphenyl)-1-pyridin-3-yl-propan-1-one (2): Asolution of olefin 1 (20.0 g, 74.2 mmol), wet 10% Pd/C (2.0 g), andammonium formate (14.0 g, 222 mmol) in MeOH (400 mL) was heated atreflux for 30 min and filtered, while hot, through a pad of Celite. Thefiltrate was allowed to slowly cool and the resulting solids werefiltered and air dried to afford product (10.0 g, 50%) as a colorlesssolid: mp 91.5-92.5° C.; TLC (EtOAc) Rf=0.55; 1H NMR (CDCl3, 300 MHz)9.16 (d, J=2.0 Hz, 1 H), 8.76 (dd, J=4.8, 1.7 Hz, 1 H), 8.21 (dt, J=8.0,1.9 Hz, 1 H), 7.40 (dd, J=7.9, 4.8 Hz, 1 H), 6.83-6.77 (m, 3 H), 3.87(s, 3 H), 3.85 (s, 3 H), 3.30 (d, J=7.3 Hz, 2 H), 3.03 (d, J=7.7 Hz, 2H); 13C NMR (CDCl3, 75 MHz) 198.2, 153.5, 149.6, 149.0, 147.6, 135.3,133.4, 132.1, 123.6, 120.2, 111.9, 111.5, 56.0 (2), 40.9, 29.5; Anal.Calcd for C16H17NO3: C, 70.83; H, 6.32; N, 5.16. Found: C, 70.63; H,6.42; N, 5.05.

[0310] (R)-3-(3,4-Dimethoxyphenyl)-1-pyridin-3-yl-propan-1-ol (3): Asolution of (+)-DIP-Chloride™ (7.09 g, 22.1 mmol) in THF (10 mL) at −25°C. was treated with ketone 2 (2.0 g, 7.37 mmol). The resulting mixturewas allowed to stand in at −20° C. for 2 h then placed in a −10° C.freezer for 48 h, after which time the mixture was concentrated andtreated with diethyl ether (50 mL) followed by diethanolamine (4.24 mL,44.2 mmol). The viscous mixture was allowed to stir at room temperaturefor 6 h after which time it was filtered through a pad of Celite withthe aid of diethyl ether. The filtrate was concentrated and the crudematerial flash chromatographed (EtOAc then 10% MeOH/EtOAc) to affordproduct. The product was redissolved in diethyl ether (50 mL) and againtreated once again with diethanolamine (2.12 mL, 22.1 mmol) as describedabove to afford product (1.74 g, 86%) as a clear colorless oil (96% eeby Chiralpak AD HPLC, 15% EtOH/hexane, retention time 6.1 min for theS-enantiomer and 19.4 min for the desired R-enantiomer): TLC (EtOAc)Rf=0.25; IR (neat) 3210, 2935, 1590, 1515, 1465, 1420, 1260, 1155, 1070,1030, 1030 cm−1; 1H NMR (CDCl3, 300 MHz) 8.50 (d, J=1.7 Hz, 1 H), 8.44

[0311] (dd, J=4.7, 1.5 Hz, 1 H), 7.71 (dt, J=7.8, 1.7 Hz, 1 H),7.28-7.24 (m, 1 H), 6.80-6.70 (m, 1 H), 4.72 (dd, J=7.9, 5.2 Hz, 1 H),3.85 (s, 6 H), 3.21 (br s, 1 H), 2.77-2.9 (m, 2 H), 2.18-1.96 (m, 2 H);13C NMR (CDCl3, 75 MHz) 149.0, 148.6, 147.7, 147.4, 140.3, 134.0, 133.8,123.6, 120.2, 111.8, 111.4, 71.3, 56.0, 55.8, 40.7, 31.5; LRMS (ES+)(M+H)+ 274; HRMS (ES+): (M+H)+ calcd: 274.1462, meas: 274.1443.

[0312]1-[2(S)-(3,4,5-trimethoxyphenyl)-butyryl]-piperdine-2(S)-carboxylicacid, 3-(3,4-Dimethoxyphenyl)-1-pyridin-3-yl-propan-1(R)-ol ester(AP22542): A solution of alcohol 3 (600 mg, 2.20 mmol), acid AP17362(882 mg, 2.42 mmol), and DMAP (2.41 mg, 1.98 mmol) in CH2Cl2 (2.5 mL) at−10° C., was treated with DCC (498 mg, 2.42 mmol). The mixture wasallowed to warm to −5° C. over a 1 h period and then placed in a 5° C.refrigerator for an additional 16 h. The reaction mixture was thendiluted with EtOAc (3 mL), filtered, evaporated, and the crude materialflash chromatographed (75% then 100% EtOAc/hexane) to afford product(1.15 g, 85%) as a colorless foam: TLC (EtOAc) Rf=0.40; IR (neat) 2940,1740, 1645, 1590, 1515, 1455, 1420, 1240, 1130, 1030 cm−1; 1H NMR(CDCl3, 300 MHz) 8.50 (dd, J=4.6, 1.5 Hz, 1 H), 8.42 (d, J=1.7 Hz, 1 H),7.27 (d, J=8.6 Hz, 1 H), 7.19 (dd, J=7.7, 4.7 Hz, 1 H), 6.78 (d, J=7.7Hz, 1 H), 6.66-6.64 (m, 2 H), 6.46 (s, 2 H), 5.69 (dd, J=7.7, 6.0 Hz, 1H), 5.47 (d, J=4.3 Hz, 1 H), 3.86-3.73 (m, 16 H), 3.59 (t, J=7.1 Hz, 1H), 2.72 (td, J=13.2, 2.6 Hz, 1 H), 2.60-2.38 (m, 2 H), 2.30 (d, J=12.4Hz, 1 H), 2.16-2.02 (m, 2 H), 1.99-1.90 (m, 1 H), 1.79-1.57 (m, 4 H),1.46-1.37 (m, 1 H), 1.32-1.19 (m, 1 H), 0.90 (t, J=7.3 Hz, 3 H); 13C NMR(CDCl3, 75 MHz) 172.6, 170.5, 153.3, 149.5, 149.0, 148.3, 147.5, 136.9,135.6, 135.3, 133.8, 1323.0, 123.6, 120.2, 111.7, 111.5, 105.1, 73.6,60.9, 56.1, 56.0, 52.0, 50.7, 43.5, 37.9, 31.1, 28.3, 26.7, 25.3, 20.9,12.5; LRMS (ES+) (M+H)+ 621; HRMS (FAB): (M+H)+ calcd: 621.3176, meas:621.3178.

[0313] Scheme 3

[0314] (R/S)-2-(3,4,5-Trimethoxyphenyl)butyric acid: A solution of of3,4,5-trimethoxyphenylacetic acid (40.0 g, 176.8 mmol) in THF (125 mL)at 0° C. was treated dropwise with a 2N THF solution of sodiumbis(trimethylsilyl)amide (181 mL, 362 mmol, Lancaster) over a 1 h periodkeeping the internal reaction temperature below 8° C. After 15 min,iodoethane (14.9 mL, 185.7 mmol) was added slowly over a 30 min periodkeeping the internal reaction temperature below 6-8° C. and the solutionallowed to warm to room temperature. After 2 h, the mixture was pouredonto EtOAc (700 mL) and acidified by slow addition of a 2.0 N HClsolution (325 mL). The organic component was further washed with asaturated sodium bisulfite solution (50 mL) followed by brine (2×50 mL),then dried over anhydrous Na2SO4, and concentrated to a waxy residue(43.8 g). The crude product was recystallized from hot EtOAc/hexane (30mL/30 mL) to afford product (37.1 g, 83%): mp 103-104° C.; TLC(AcOH/EtOAc/hexane, 2:49:49) Rf=0.50.

[0315] (S)-2-(3,4,5-Trimethoxyphenyl)butyric acid (4S): A solution of 4(3.09 g, 12.15 mmol) in CH3CN (130 mL) was treated with (−)-cinchonidine(3.58 g, 12.15 mmol) and the mixture heated to reflux. The homogeneoussolution was allowed to slowly cool to room temperature with concomitantformation of salts. After a period of 1 h at room temperature, thesolution was cooled to 0° C. for 30 minutes and the solution thenfiltered to afford 4.05 g of a chalky colorless solid. Thisrecrystalliztion procedure was then carried out an addition four timesutilizing −20 mL CH3CN/g of salt. The diastereomeric salt isolated fromthe fifth crystallization (1.64 g) was suspended in EtOAc (100 mL) andtreated with a 10% aqueous HCl solution (10 mL). The organic phase wasthen washed with water (2×15 mL) followed by brine 10 mL), dried overanhydrous MgSO4, and concentrated to afford product (0.75 g, 24%) as acolorless solid (91% ee by Chiralcel OD HPLC, 1:5:94 formicacid/i-PrOH/hexane, retention time 19.6 min for the R-enantiomer, and22.1 min for the desired S-enantiomer): mp 84-85° C. (99.1% eematerial); [a]22D +54.8 (c=1.07, MeOH, 30 min, 99.1% ee material); UV(MeOH) lmax 270 (e 895), 232 (e 7,440), 207 (e 40,994) nm; 1H NMR(DMSO-d6, 300 MHz) 6.34 (s, 2 H), 3.52 (s, 6 H), 3.40 (s, 3 H), 3.11 (t,J=7.6 Hz, 1 H) 1.76-164 (m, 1 H), 1.46-1.36 (m, 1 H), 0.60 (t, J=7.3 Hz,3 H); 1H NMR (CD3OD, 300 MHz) 6.78 (s, 2 H), 4.00 (s, 6 H), 3.90 (s, 3H), 3.55 (t, J=7.7 Hz, 1 H) 2.24-2.12 (m, 1 H), 1.97-1.83 (m, 1 H), 1.07(t, J=7.3 Hz, 3 H); 13C NMR (DMSO-d6, 75 MHz) 175.1, 153.1, 136.9,135.8, 105.4, 60.3, 56.2, 53.1, 26.7, 12.4; 13C NMR (CD3OD, 75 MHz)178.1, 154.9, 138.7, 137.4, 106.8, 61.5, 57.0, 55.3, 28.3, 12.9; HRMS(FAB): (M−H)− calcd: 253.1076, meas: 253.1063. Anal. Calcd for C13H1805:C, 61.41; H, 7.13. Found: C, 61.47; H, 7.20.

[0316][S-(R*,R*)]-1-[1-oxo-2-(3,4,5-trimethoxyphenyl)butyl]-2-piperdinecarboxylicacid (AP17362): A solution of 5 (0.75 g, 2.95 mmol, 91% ee) in CH2Cl2(15 mL) was treated with methyl-L-pipecolate hydrochloride (0.539 g,3.00 mmol) followed by 2-chloro-1-methylpyridinium iodide (0.958 g, 3.75mmol) and triethylamine (1.25 mL, 8.95 mmol). The reaction mixture wasallowed to stir for 3.5 h, after which time the solution was dilutedwith EtOAc (100 mL), washed with water (15 mL), a 5% aqueous citric acidsolution (25 mL), a saturated Na2CO3 solution (10 mL), water (15 mL),and finally brine (15 mL). The organic phase was dried over MgSO4 andconcentrated to a yellow oil which was then dissolved in MeOH (14 mL).The methanolic solution was treated with water (1 mL) followed bylithium hydroxide monohydrate (0.620 g, 14.78 mmol). After 4 h, themixture was diluted with EtOAc (100 mL), washed with a saturated NaHCO3solution (3×40 mL) followed by water (20 mL). The aqueous portions werecombined and acidified to pH ˜3 by careful addition of a 10% aqueous HClsolution. The resulting suspension was extracted with EtOAc (2×75 mL)which was then washed with water (2×25 mL), brine (20 mL), dried overMgSO4, and concentrated to a solid which was dissolved in a refluxingEtOAc (75 mL) solution and allowed to slowly cool to room temperature.The resulting crystalline material was filtered and air dried to affordproduct (0.508 g, 42%) as a colorless solid: (+99% de by Chiralpak ADHPLC with guard column, 0.2:5:95 formic acid/i-PrOH/hexane, retentiontime 40.0 min for the SR-diastereomer, 43.0 min for the desiredSS-diastereomer, 46.5 min for the RR-diastereomer, and 67.5 min for theRS-diastereomer); mp 173.5-174° C.; [a]22D +10.9 (c=1.01, DMSO, 30 min);UV (MeOH) lmax 270 (e 990), 232 (e 11,161), 207 (e 49,079) nm; 1H NMR(DMSO-d6, 300 MHz) 6.55 (s, 2 H), 5.13 (d, J=4.4 Hz, 1 H), 3.85-3.64 (m,11 H), 2.77-2.70 (m, 1 H), 2.12 (d, J=13.4 Hz, 1 H), 1.99-1.85 (m, 1 H),1.65-1.55 (m, 4 H), 1.38-1.18 (m, 2 H), 0.84 (t, J=7.2 Hz, 3 H); 1H NMR(CD3OD, 300 MHz) 6.74 (s, 2 H), 5.43 (d, J=4.0 Hz, 1 H), 4.13-3.83 (m,11 H), 3.03 (td, J=13.5, 3.0 Hz, 1 H), 2.44 (d, J=13.8 Hz, 1 H),2.24-2.14 (m, 1 H), 1.90-1.40 (m, 6 H) 1.09 (t, J=7.3 Hz, 3 H); 13C NMR(DMSO-d6, 75 MHz) 172.9, 172.2, 153.0, 136.2, 105.4, 60.2, 56.2, 56.0,51.8, 49.4, 43.1, 28.5, 26.8, 25.3, 21.0, 12.8; 13C NMR (CD3OD, 75 MHz)175.4, 174.5, 154.9, 137.5, 106.8, 61.5, 57.1, 53.9, 52.1, 45.2, 29.9,28.2, 26.8, 22.3, 13.2; HRMS (FAB): (M−H)− calcd: 364.1760, meas:364.1774. Anal. Calcd for C19H27O6: C, 62.45; H, 7.45; N, 3.83. Found:C, 62.32; H, 7.61; N, 3.88.

Example 3

[0317] The Conditional Retention Domain F(36M) FKBP; Studies with hGH

[0318] To test whether F(36M) could function as a conditional retentiondomain to enable regulated secretion of a fused heterologous protein, afusion protein of the design shown in FIG. 2B was constructed. Thisfusion protein contains a signal sequence from the human growth hormone(hGH) gene, 4 copies of the F(36M) domain, a furin cleavage sequencefrom human stromelysin 3 and coding sequence from the mature hGHprotein. The resulting fusion protein, in essence, simply containsF(36M) domains and a furin cleavage signal inserted at the cleavage sitebetween the signal sequence and the mature hGH peptide sequence. Sincethe furin recognition sequence is N-terminal to the cleavage site it canbe situated so that appropriate cleavage will generate the same hGHamino acid sequence as that generated by natural cleavage of its ownsignal sequence.

[0319] A vector driving the expression of this fusion protein, undercontrol of the strong constitutive enhancer from CMV, was transientlytransfected into HT1080 cells, a human fibrosarcoma cell line. Followingovernight incubation of cells in the absence of ligand, the medium waswashed away and fresh medium added, with or without ligand. Two hourslater, the amount of hGH secreted into the medium was determined byradioimmunoassay. As shown in FIG. 3A, in the absence of ligand, theamount of hGH secreted was low. In contrast, in the presence of ligand,the amount of hGH secreted was several hundred-fold greater. Thisdemonstrates that F(36M) can act as a conditional retention domain whenfused to a heterologous protein.

[0320] Next, cell lines were generated by stably transfecting theF(36M)-hGH expression vector into HT1080 cells. For comparison, thenative hGH gene driven by the same CMV enhancer was also stablytransfected into cells. To allow an initial assessment of any potentialtoxic effects of the retained fusion protein, the selectable marker wasexpressed from the same transcript as the wt hGH or F(36M)-hGH fusionproteins through the use of an internal ribosome entry signal.Equivalent numbers of clones were obtained, suggesting the there was notoxic effect of the fusion protein.

[0321] Pools of clones stably transfected with the F(36M)-hGH fusionprotein (HT88 pool) were analyzed as described for the transientlytransfected cells. As shown in FIG. 3B, once again, hGH secretion, whichis very low in the absence of ligand, is induced several hundred fold byincubation with ligand for two hours. To determine the constitutive rateof hGH secretion, the amount of hGH secreted in the presence of ligandwas measured from cells that had already been exposed to ligand for 15hours. As shown in lane 3, the constitutive rate of secretion from theHT88 cell line was very similar to the rate of secretion from the HT89cell line which had been stably transfected with the wild type hGHprotein (lane 4). Thus, in the presence of ligand the F(36M) domainshave no detectable “retention” activity. Furthermore, this shows that inthe absence of ligand, the fusion protein accumulates to levelsapproximately 10-fold higher than that seen when secretion is notblocked. This steady state level of stored F(36M)-hGH fusion protein canpersist for months in the cell line with no apparent toxic effect.

Example 4

[0322] Localization/cleavage of Fusion Protein

[0323] To analyze the localization of the fusion protein, EGFP codingsequence was incorporated in the fusion protein as shown in FIG. 2C. Incells stably transfected with this fusion protein, in the absence ofligand, the fusion protein was visible as large green spots concentratedat multiple points in the perinuclear space. Co-localization experimentsdemonstrated that the fusion protein is aggregated and retained withinthe ER, as predicted (J. Rothman, data not shown). Upon addition ofligand, the aggregates disperse over the next 15 to 60 minutes. Thisdisaggregation coincides with the appearance of hGH protein in thesupernatant of the cells. As described for the HT88 cell line, ligandinduces a several hundred fold increase in hGH (data not shown).

[0324] To analyze further the state of the fusion protein, cell lysatesand supernatants were prepared from the HT88 cells that had beenincubated in the presence or absence of ligand for 2 hours. Thesesamples were then immunoblotted with anti-hGH and anti-FKBP antibodies.As shown in FIG. 4, in the absence of ligand an approximately 75kDa-sized band, which corresponds to the expected size of the F(36M)-hGHfusion protein, is detected in the lysate (lane 1) but not thesupernatant (lane 3) of unstimulated cells with both the anti-hGH andanti-FKBP antibodies. In cells that had been stimulated with ligand for2 hours, very little fusion protein is detected in the cell lysate, butinstead, cleaved proteins are detected in the supernatant. The anti-hGHblot shows the presence of a 22 kDa sized protein (lane 6) thatco-migrates with purified recombinant hGH (lane 7). The anti-FKBP blotshows the presence of a 53 kDa protein that is around the expected sizeof the remainder of the fusion protein (F(36M)-FCS). Together theseresults indicate that the F(36M)-hGH fusion protein is indeed retainedwithin the ER in the absence of ligand, released upon interaction withligand and subsequently cleaved at the appropriate position, resultingin the secretion of the F(36M)-FCS portion of the fusion protein and anintact hGH protein.

Example 5

[0325] Dose Response and Kinetics of hGH Secretion

[0326] The amount of hGH secreted from HT88 cells in response to ligandis dose-dependent (FIG. 5A). Peak level of secretion occurs atapproximately 2-3 uM AP21998 with half-maximal secretion occurring at600 nM.

[0327] To determine the kinetics of secretion, cells were stimulatedwith ligand and an aliquot of medium collects at various time points tomeasure the accumulation of hGH in the supernatant. Following additionof saturating levels of ligand, low levels of hGH are detected in thesupernatant within minutes with the peak rate of secretion occurringbetween 20-30 minutes (FIG. 5B). This corresponds to the amount of timeit takes for a newly synthesized protein to be secreted. After the bolusrelease of stored fusion protein, the rate of secretion rapidlydecreases.

[0328] To further examine the kinetics of secretion in response toligand, cells were incubated overnight in the presence or absence ofligand and then medium collected at 1 hour intervals. The cells werewashed extensively between time points and the medium replaced withmedium containing or lacking ligand as indicated. FIG. 6A (group A)shows the constitutive rate of secretion from the cells. In group B, alarge bolus release is observed since the cells had not been exposed toligand previously. Once ligand is washed away, however, the rate of hGHsecretion quickly decreases, returning to the low basal rate within 2hours. Group C shows that if the cells are exposed to ligand followingthe large bolus release, within 2 hours the rate of secretion matchesthat of the constitutively producing cells (group A).

[0329] Since the constitutive rate of hGH production is only about 75ng/million cells/hr while 1250 ng/million cells is released in the firsthour after the stores are emptied, it should take some time for thestores to be refilled. As shown in FIG. 6B, when the stored hGH isreleased by incubation with maximal concentration of ligand, it takesbetween 8-24 hours for the stores to be refilled so that the magnitudesecond bolus release matches that of the first (or exceeds it since thecell number has increased in the time). Therefore, in order to achieveconsistent, rapid, pulsatile secretion the stores must not be emptiedcompletely. As shown in FIG. 6c, if sub-maximal concentrations of ligandare added (e.g. 250 or 500 nM), an equivalent amount of hGH can besecreted 4 hours later.

[0330] The degree of aggregation increases as the number of F(36M)domains increases. To test whether the degree of retention could bemanipulated, constructs containing 2, 3 or 6 F(36M) domains were fusedto hGH, stable cell lines generated and hGH secreted in the presence andabsence of ligand assayed. As shown in FIG. 7, the basal secretion inthe absence of ligand increases as the number of F(36M) domainsdecreases. This likely reflects a reduction in the size of aggregateswhich permits monomeric fusion proteins to escape retention. An increasein the “leakiness” of fusion protein secretion is also reflected as adecrease in the amount of stored fusion protein and, hence, the amountof protein released in bolus upon addition of ligand. It may be possibleto exploit this to provide transient high level induced secretionagainst a back drop of relatively high constitutive basal secretion.Such a situation may be particularly desirable in the case of insulinproduction for the treatment of type 1 diabetes.

Example 6

[0331] Regulated Insulin Secretion

[0332] To test whether the conditional retention domain, F(36M), couldalso be used to enable regulated secretion of insulin, a fusion proteinof the design shown in FIG. 3D was constructed. This fusion protein isanalogous to the F(36M)4-hGH fusion protein described in example X,except the mature hGH coding sequence has been replaced by codingsequence from the mature human insulin gene. Normally, in islet cells,proinsulin is processed into the mature, active, A and B chain complexby endopeptidases that are expressed exclusively in neuroendocrinecells. Therefore, to allow insulin to be processed properly innon-endocrine cells mutations were introduced at the B-C and C-Ajunctions that would allow processing by the ubiquitous protease furin(Groskreutz et al., J. Biol. Chem. 269:6241-6245, 1994). In addition, athird mutation, in which amino acid 10 of the B chain (histidine) wasmutated to aspartic acid, was introduced to increase the stability ofthe protein (Groskreutz et al., J. Biol. Chem. 269:6241-6245 1994).

[0333] A vector driving expression of this F(36M)-insulin fusion protein(F(36M)4-hIn-m3) was transiently transfected into HT1080 cells. Forcomparison, vectors driving the expression of insulin protein alone,with (hIn-m3) or without (hIn-wt) the three mutations were alsotransfected. Following overnight incubation of cells in the absence ofligand, the medium was washed away and fresh medium added, without orwith increasing concentrations of the monomeric ligand, AP21998. Threehours later, the amount of insulin secreted into the medium wasdetermined by ELISA using an assay that recognizes an epitope within theC-peptide (ALPCO). As shown in FIG. 8, fusion of F(36M) domains toinsulin results in a suppression of its secretion in the absence ofmonomeric ligand. Furthermore, secretion is induced in the presence ofmonomer in a dose-dependent manner.

Example 7

[0334] Regulated Expression of a Membrane Tethered Protein

[0335] To determine whether the CRD, F(36M), could also be used toregulate surface expression of a membrane-tethered protein, 3, 4, or 6copies of F(36M) were fused to the extracellular and transmembraneportions of the low-affinity nerve growth factor receptor (LNGFR; FIG.3E). In these fusion proteins the F(36M) domains should be localized tothe cytoplasm and tethered to the plasma membrane, in contrast to thehGH and insulin fusions described in examples 3 and 6, in which theF(36M) domains were expressed as part of a soluble protein thatlocalized initially to the lumen of the ER. Surface expression wasassessed by FACS analysis using anti-LNGFR antibodies (Chromaprobe,Mountain View, Calif.). As shown in FIG. 9, upon transfection intoHT1080 cells two peaks, corresponding to low and high levels, of LNGFRsurface expression are detected in the absence of monomer with eachfusion protein. The relatively high level of surface expression in theabsence of monomer suggests that the retention activity of the F(36M)domains is not as strong when the fusion protein is tethered to themembrane, compared to when it is in solution. This may reflect thepresence of physical constraints that prevent formation of high orderoligomers. However, these results show that the retention activity ofthe F(36M) domains clearly increases as the number of F(36M) domainsincrease. Furthermore, in the presence of monomeric ligand, surfaceexpression increases significantly in all cases. Thus F(36M) domains canalso be used to conditionally induce surface expression of amembrane-tethered protein.

Example 8

[0336] Construction and Testing of a Construct for Conditional Secretionof hGH Using Rat Retinol Binding Protein as a CRD

[0337] Rat retinol binding protein (rRBP) is conditionally retained inthe ER of a variety of cell types unless retinol is added (Melhus etal., J Biol Chem 1992 vol 26712036-12041), and so is a suitablecandidate for use as a CRD. We assembled a construct to test whetherrRBP could be used to obtain conditional secretion of the target proteinhuman growth hormone (hGH) in response to retinoid ligands. The generalstructure of the construct is shown below:

[0338] The construct comprises the rRBP cDNA, including the authenticsignal sequence (SS), followed by sequence encoding the furin cleavagesite (FCS) derived from stromelysin E (the amino acid sequence SARNRQKR(SEQ ID NO. 1) and then the mature 191 amino acid cDNA coding sequenceof hGH (lacking the signal sequence) followed by an in-frame stop codon.The stromelysin E cleavage site was chosen because it is of human origin(and therefore expected to be minimally inuntmogenic in future humantherapeutic applications), and because it is known to be recognised byfurin in the context of fusion to proteins where the P1′ residue—theresidue following the cleavage site—is Phe, as in hGH (for a review seeDenault and Leduc, FEBS Lett 1996 vol 379, 113-116). All junctionsbetween the various sequence motifs and domains are direct and includeno additional sequence, with the exception of an additional threoninecodon between rRBP and FCS to accommodate the SpeI site. The expressioncassette was cloned into the expression vector pC4EN, placing expressionunder the control of the strong hCMV immediate early promoter andenhancer.

[0339] A DNA fragment encompassing the rRBP cDNA was obtained by RT-PCRfrom rat liver poly A+RNA (obtained from Clontech, catalog # 6710-1)using the Clontech first strand kit with random primers, followed by PCRunder conventional amplification conditions using primers RBP-5′ (263)and RBP-3′ (264). The PCR product was purified and digested with EcoRIand SpeI. A second DNA fragment encoding the FCS and mature hGH codingsequence was obtained by PCR amplification from the hGH cDNA expressionvector Z12IHB. The PCR primers used were FCS-hGH-5′ (265) and hGH-3′(266); primer FCS-hGH-5′ (265) includes additional sequence that encodesthe FCS. The PCR product was purified and digested with SpeI and BamHi.The two DNA fragments were then cloned into EcoRI-BamHI-opened pC4EN ina three-way ligation to produce the final expression vectorpC4EN-rRBP-hGH. Positive clones were completely sequenced to check thatno errors were incorporated during cloning.

[0340] The construct contains restriction sites that can be used to addadditional modules to the expressed fusion protein. Thus the stromelysinE FCS can be replaced with alternative cleavage sites by excising theexisting SpeI-AflII fragment and cloning in an appropriate SpeI-AflIIcompatible oligonucleotide pair. An epitope tag can be appended to therRBP, upstream of the FCS, to allow immunochemical tracking of the rRBPmodule inside cells. Alternative target proteins can be cloned asSpeI-XmaI fragments (the use of the 3′ BamHI site is precluded by theexistence of another BamHI site in the rRBP coding sequence).Alternative CRDs can be cloned in place of rRBP as EcoRI-SpeI fragments.

[0341] Particularly important additional constructs are those thatincorporate multiple reiterated copies of rRBP. These are obtained byreamplifying pC4EN-rRBP-hGH using primers 5′-RBP-Xba and 3′-RBP-Spe,generating a fragment containing the mature rRBP sequence (no signalsequence) flanked by SpeI-compatible 5′ XbaI and 3′ SpeI sites. The PCRproduct is purified, digested with XbaI and SpeI, and cloned intoSpeI-opened pC4EN-rRBP-hGH to generate pC4EN-(rRBP×2)-hGH. An analogousprocedure can be used to prepare constructs encoding higher orderconcatenates of rRBP.

[0342] PCR Primers:

[0343] RBP-5′ (263) 5′ CGTACgaattcCAGAAGCGCGTATGGAGTGGGTGTGGGCGCTCGTGCTG(SEQ ID NO. 23)

[0344] RBP-3′ (264) 5′GCATGactagtCAAACTGTTTCTTGAGGGTCTGCTTTGACAG (SEQ IDNO. 24)

[0345] FCS-hGH-5′ (265) (SEQ ID NO. 25)

[0346]5′GCAACactagtGCTAGAAACCGTCAGAAGAGATTCCCAACCATTCCCTTAAGCAGGCCTTTTGACAACGC(SEQ ID NO. 26)

[0347] hGH-3′ (266) 5′GCTCAggatccCGGGCTAGAAGCCACAGCTGCCCTCCACAGAGCG (SEQID NO. 27)

[0348] 5′-RBP-Xba 5′TCAGCtctagaGAGCGCGACTGCAGGGTGAGC (SEQ ID NO. 28)

[0349] 3′-RBP-Spe 5′GAAGCactagtCAAACTGTTTCTTGAGGGTCTG (SEQ ID NO. 29)

[0350] The sequence of the expression cassette is as follows (keyrestriction sites underlined): EcoRI             rRBP signal sequence-->(SEQ ID NO. 30) 1 gaattccagaagcgcgt ATG GAG TGG GTG TGG GCG CTC GTG CTGCTG GCG GCT CTG GGA GGC 62 (SEQ ID NO. 31) 1                  M   E   W   V   W   A   L   V   L   L   A   A   L   G   G15              rRBP mature protein sequence--> 63 GGC AGC GCC GAG CGCGAC TGC AGG GTG AGC AGC TTC AGA GTC AAG GAG AAC TTC GAC AAG 122 16G   S   A   E   R   D   C   R   V   S   S   F   R   V   K   E   N   F   D   K35                                                   BamHI 123 GCT CGTTTC TCT GGG CTC TGG TAT GCC ATC GCC AAA AAG GAT CCC GAG GGT CTC TTT TTG182 36A   R   F   S   G   L   W   Y   A   I   A   K   K   D   P   E   G   L   F   L55 183 CAA GAC AAC ATC ATC GCT GAG TTT TCT GTC GAC GAG AAG GGT CAT ATGAGC GCT ACA GCC 242 56Q   D   N   I   I   A   E   F   S   V   D   E   K   G   H   M   S   A   T   A75 243 AAG GGA CGA GTC CGT CTT CTG AGC AAC TGG GAA GTG TGT GCA GAC ATGGTG GGC ACT TTC 302 76K   G   R   V   R   L   L   S   N   W   E   V   C   A   D   M   V   G   T   F95 303 ACA GAT ACA GAA GAT CCT GCC AAG TTC AAG ATG AAG TAC TGG GGT GTAGCC TCC TTT CTC 362 96T   D   T   E   D   P   A   K   F   K   M   K   Y   W   G   V   A   S   F   L115 363 CAG CGA GGA AAC GAT GAC CAC TGG ATC ATC GAT ACG GAC TAC GAC ACCTTC GCT CTG CAG 422 116Q   R   G   N   D   D   H   W   I   I   D   T   D   Y   D   T   F   A   L   Q135 423 TAT TCC TGC CGC CTG CAG AAT CTG GAT GGC ACC TGT GCA GAC AGC TACTCC TTT GTG TTT 482 136Y   S   C   R   L   Q   N   L   D   G   T   C   A   D   S   Y   S   F   V   F155 483 TCT CGT GAC CCC AAT GGC CTG ACC CCG GAG ACA CGG AGG CTG GTG AGGCAG CGA CAG GAC 542 156S   R   D   P   N   G   L   T   P   E   T   R   R   L   V   R   Q   R   Q   E175 543 GAG CTG TGC CTA GAG AGG CAG TAC AGA TGG ATC GAG CAC AAT GGT TACTGT CAA AGC AGA 602 176E   L   C   L   E   R   Q   Y   R   W   I   E   H   N   G   Y   C   Q   S   R195                       Spel  FCS-->                          maturehGH--> 603 CCC TCA AGA AAC AGT TTG ACT AGT GCT AGA AAC CGT CAG AAG AGATTC CCA ACC ATT CCC 662 196P   S   R   N   S   L   T   S   A   R   N   R   Q   K   R   F   P   T   I   P215 AflII 663 TTA AGC AGG CCT TTT GAC AAC GCT ATG CTC CGC GCC CAT CGTCTG CAC CAG CTG GCC TTT 722 216L   S   R   P   F   D   N   A   M   L   R   A   H   R   L   H   Q   L   A   F235 723 GAC ACC TAC CAG GAG TTT GAA GAA GCC TAT ATC CCA AAG GAA CAG AAGTAT TCA TTC CTG 782 236D   T   Y   Q   E   F   E   E   A   Y   I   P   K   E   Q   K   Y   S   P   L255 783 CAG AAC CCC CAG ACC TCC CTC TGT TTC TCA GAG TCT ATT CCG ACA CCCTCC AAC AGG GAG 842 256Q   N   P   Q   T   S   L   C   F   S   E   S   I   P   T   P   S   N   R   E275 843 GAA ACA CAA CAG AAA TCC AAC CTA GAG CTG CTC CGC ATC TCC CTG CTGCTC ATC CAG TCG 902 276E   T   Q   Q   K   S   N   L   E   L   L   R   I   S   L   L   L   I   Q   S295 903 TGG CTG GAG CCC GTG CAG TTC CTC AGG AGT GTC TTC GCC AAC AGC CTGGTG TAC GGC GCC 962 296W   L   E   P   V   Q   F   L   R   S   V   F   A   N   S   L   V   Y   G   A315 963 TCT GAC AGC AAC GTC TAT GAC CTC CTA AAG GAC CTA GAG GAA GGC ATCCAA ACG CTG ATG 1022 316S   D   S   N   V   Y   D   L   L   K   D   L   E   E   G   I   Q   T   L   M335 1023 GGG AGG CTG GAA GAT GGC AGC CCC CGG ACT GGG CAG ATC TTC AAG CAGACC TAC AGC AAG 1082 336G   R   L   E   D   G   S   P   R   T   G   Q   I   F   K   Q   T   Y   S   K355 1083 TTC GAC ACA AAC TCA CAC AAC GAT GAC GCA CTA CTC AAG AAC TAC GGGCTG CTC TAC TGC 1142 356F   D   T   N   S   H   N   D   D   A   L   L   K   N   Y   G   L   L   Y   C375 1143 TTC AGG AAG GAC ATG GAC AAG GTC GAG ACA TTC CTG CGC ATC GTG CAGTGC CGC TCT GTG 1202 376F   R   K   D   M   D   K   V   E   T   F   L   R   K   V   Q   C   R   S   V395 1203 GAG GGC AGC TGT GGC TTC TAGcccgggatcctgagaacttcagggtgagtttggggacccttgattgttcttt 1275 396E   G   S   C   G   F   *        BamHI 402

[0351] The sequence of the encoded protein is as follows: (SEQ ID NO.31) MEWVWALVLLAALGGGSAERDCRVSSFRVKENFDKARFSGLWYAIAKKDPEGLFLQDNIIAEFSVDEKGHMSATAKGRVRLLSNWEVCADMVGTFTDTEDPAKFKMKYWGVASFLQRGNDDHWIIDTDYDTFALQYSCRLQNLDGTCADSYSFVFSRDPNGLTPETRRLVRQRQEELCLERQYRWIEHNGYCQSRPSRNSLTSARNRQKRFPTIPLSRPFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCG F

[0352] The nucleotide sequence of the expression cassette is as follows:(SEQ ID NO. 32)ATGGAGTGGGTGTGGGCGCTCGTGCTGCTGGCGGCTCTGGGAGGCGGCAGCGCCGAGCGCGACTGCAGGGTGAGCAGCTTCAGAGTCAAGGAGAACTTCGACAAGGCTCGTTTCTCTGGGCTCTGGTATGCCATCGCCAAAAAGGATCCCGAGGGTCTCTTTTTGCAAGACAACATCATCGCTGAGTTTTCTGTCGACGAGAAGGGTCATATGAGCGCTACAGCCAAGGGACGAGTCCGTCTTCTGAGCAACTGGGAAGTGTGTGCAGACATGGTGGGCACTTTCACAGATACAGAAGATCCTGCCAAGTTCAAGATGAAGTACTGGGGTGTAGCCTCCTTTCTCCAGCGAGGAAACGATGACCACTGGATCATCGATACGGACTACGACACCTTCGCTCTGCAGTATTCCTGCCGCCTGCAGAATCTGGATGGCACCTGTGCAGACAGCTACTCCTTTGTGTTTTCTCGTGACCCCAATGGCCTGACCCCGGAGACACGGAGGCTGGTGAGGCAGCGACAGGAGGAGCTGTGCCTAGAGAGGCAGTACAGATGGATCGAGCACAATGGTTACTGTCAAAGCAGACCCTCAAGAAACAGTTTGACTAGTGCTAGAAACCGTCAGAAGAGATTCCCAACCATTCCCTTAAGCAGGCCTTTTGACAACGCTATGCTCCGCGCCCATCGTCTGCACCAGCTGGCCTTTGACACCTACCAGGAGTTTGAAGAAGCCTATATCCCAAAGGAACAGAAGTATTCATTCCTGCAGAACCCCCAGACCTCCCTCTGTTTCTCAGAGTCTATTCCGACACCCTCCAACAGGGAGGAAACACAACAGAAATCCAACCTAGAGCTGCTCCGCATCTCCCTGCTGCTCATCCAGTCGTGGCTGGAGCCCGTGCAGTTCCTCAGGAGTGTCTTCGCCAACAGCCTGGTGTACGGCGCCTCTGACAGCAACGTCTATGACCTCCTAAAGGACCTAGAGGAAGGCATCCAAACGCTGATGGGGAGGCTGGAAGATGGCAGCCCCCGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGATGACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCTTCTAG

[0353] To test whether fusion to rRBP can result in ligand-dependentprevention of secretion of hGH, pC4EN-rRBP-hGH, pC4EN-(rRBP×2)-hGH andtheir derivatives are transiently transfected into the humanfibrosarcoma cell line HT1080 using standard methods (eg see Rivera etal., Nature Med 2: 1028-1032 1996; Amara et al., PNAS 94:10618-106231997). After overnight incubation, medium is removed and new mediumadded, containing either no drug or retinol at various concentrations.After a further incubation of 2-24 hours, the amount of hGH secretedinto the medium is determined by radioimmunoassay (Rivera et al., NatureMed 2: 1028-1032 1996).

[0354] A critical feature for these experiments, as described by Melhuset al. (J Biol Chem 1992 vol 26712036-12041), is the use of delipidizedserum in the culture medium, since untreated serum contains significantamounts of retinoids that might lead to secretion of rRBP in the absenceof exogenously added retinol. Methods for preparing delipidized serumare known (Rothblat et al., In Vitro 1976 vol 12, 554-557).

[0355] Increased secretion of hGH upon addition of increasingconcentrations of retinol would indicate that rRBP is acting as a CRD toretain hGH in secretory compartments until addition of retinol.Experiments to identify the best configuration of the system includeengineering multimers of rRBP to attempt to enhance the retentioneffect, and testing of a variety of different retinol analogs foractivity. Further experiments to confirm the subcellular location ofrRBP fusions include immunocytochemical subcellular localization ofcomponents of the constructs before and after addition of retinoidsusing, for example, anti-hGH or anti-rRBP antibodies.

Example 9

[0356] Physiological Effects of Regulated Insulin Secretion in vivo

[0357] To test whether this system could be used to regulate secretionof insulin in vivo and effect changes in serum glucose levels, 2×10e7HT101-10p cells were implanted intramuscularly into male nu/nu mice.HT101-10p cells were generated by stably transfecting HT1080 cells witha vector that drives expression of the F(36M)4- hIn-m3 fusion protein.Mice were made hyperglycemic by treatment 2 days earlier with 300 mg/kgstreptozotocin (STZ). As shown in FIG. 11a, STZ treatment elevates serumglucose levels to −350 mg/dl from −100 mg/dl seen in non-STZ treatedmice. Approximately 1 hr after cells are implanted, animal receivedvehicle or the indicated dose of intravenous AP22542 (an analog ofAP21998). Two hours later, serum samples were collected and assayed forinsulin (Ultrasensitive human insulin-specific RIA, Linco) and glucose(Sigma) concentrations. As shown in FIG. 11a, treatment of hyperglycemicmice with vehicle or a low dose of AP22542 (1 mg/kg) fails to increaseserum insulin levels above the lower limit of detection and there is nochange in serum glucose. In contrast, in animals treated with 10 mg/kgAP22542, serum insulin levels increase to −200 pM and serum glucoselevels decline to −75 mg/dl.

[0358] To examine the kinetics of this ligand-induced reduction in serumglucose, STZ-treated mice implanted with 2×10e7 HT101-10p cells wereadministered a single dose of 30 mg/kg AP22542 intravenously. Serumglucose levels were measured at various times between 5 minutes and 10hours later. As shown in FIG. 11b, at 5 and 15 minutes afteradministration of AP22542, serum glucose levels are indistinguishablefrom animals treated with vehicle. However, within 30 minutes there is asignificant reduction in serum glucose and by two hours serum glucoselevels have declined to 50 mg/dl from initial levels of nearly 500mg/dl. This effect is transient as serum glucose levels rise to 350mg/dl within 5 hours and return to baseline between 6 and 10 hourslater. Since insulin secretion is dependent on the presence of the drug,administration of lower doses of AP22542 or of a ligand with a shorterhalf life should result in an even more transient production of secretedprotein and resulting physiological effect. Conversely, administrationof higher doses of AP22542 or of a ligand with a longer half life shouldresult in a more prolonged production of secreted protein.

1 38 1 7 PRT Homo sapiens 1 Met Ser Met Arg Val Arg Arg 1 5 2 7 PRT Homosapiens 2 Lys Pro Ala Lys Ser Ala Arg 1 5 3 6 PRT Homo sapiens 3 Lys SerVal Lys Lys Arg 1 5 4 7 PRT Homo sapiens 4 Ala Arg Asn Arg Gln Lys Arg 15 5 7 PRT Homo sapiens 5 Arg Pro Ser Arg Lys Arg Arg 1 5 6 7 PRT Homosapiens 6 Thr Glu Lys Arg Lys Lys Arg 1 5 7 40 DNA Artificial SequenceDescription of Artificial SequencePCR primer 7 tcccgcacct cttcggccagcgaattccag aagcgcgtat 40 8 32 DNA Artificial Sequence Description ofArtificial SequencePCR primer 8 gactcactat aggacgcgtt cgagctcgcc cc 32 931 DNA Artificial Sequence Description of Artificial SequencePCR primer9 catcattttg gcaaaggatt cactcctcag g 31 10 27 DNA Artificial SequenceDescription of Artificial SequencePCR primer 10 gatggaaaga aaatggattcctcccgg 27 11 24 DNA Artificial Sequence Description of ArtificialSequencePCR primer 11 tctagagtga gcaagggcga ggag 24 12 37 DNA ArtificialSequence Description of Artificial SequencePCR primer 12 ggatccttattaactagtct tgtacagctc gtccatg 37 13 25 DNA Artificial SequenceDescription of Artificial SequencePCR primer 13 aagcttacca ctcagggtcctgtgg 25 14 19 DNA Artificial Sequence Description of ArtificialSequencePCR primer 14 gaattcgtgg caacttcca 19 15 29 DNA ArtificialSequence Description of Artificial SequencePCR primer 15 cacaggaccctgaattctaa gcttgtggc 29 16 30 DNA Artificial Sequence Description ofArtificial SequencePCR primer 16 ataagggaat ggttctagag gcactgccct 30 1731 DNA Artificial Sequence Description of Artificial SequencePCR primer17 atgccacccg ggactagtga agccacagct g 31 18 48 DNA Artificial SequenceDescription of Artificial SequencePCR primer 18 actagtgcta gaaaccgtcagaagagattc ccaaccattc ccttaagc 48 19 31 DNA Artificial SequenceDescription of Artificial SequencePCR primer 19 ggatcccggg ctagaagccacagctgccct c 31 20 32 DNA Artificial Sequence Description of ArtificialSequencePCR primer 20 cgaattcttc tgccatggcc ctgtggatgc gc 32 21 31 DNAArtificial Sequence Description of Artificial SequencePCR primer 21cggatccgca ggctgcgtct agttgcagta g 31 22 52 DNA Artificial SequenceDescription of Artificial SequencePCR primer 22 cactagtgct agaaaccgtcagaagagatt tgtgaaccaa cacctgtgcg gc 52 23 31 DNA Artificial SequenceDescription of Artificial SequencePCR primer 23 cggatccgca ggctgcgtctagttgcagta g 31 24 27 DNA Artificial Sequence Description of ArtificialSequencePCR primer 24 cctgtgcggc tcagacctgg tggaagc 27 25 35 DNAArtificial Sequence Description of Artificial SequencePCR primer 25cttctacaca cccaggacca agcgggaggc agagg 35 26 29 DNA Artificial SequenceDescription of Artificial SequencePCR primer 26 ccctggaggg gtcccggcagaagcgtggc 29 27 341 DNA Homo sapiens 27 ctgggggcct tgcttggcaa cagcacagacccagctgtgt tcacagacct ggcatccgtc 60 gacaactccg agtttcagca gctgctgaaccagggcatac ctgtggcccc ccacacaact 120 gagcccatgc tgatggagta ccctgaggctataactcgcc tagtgacagg ggcccagagg 180 ccccccgacc cagctcctgc tccactgggggccccggggc tccccaatgg cctcctttca 240 ggagatgaag acttctcctc cattgcggacatggacttct cagccctgct gagtcagatc 300 agctcctaag ggggtgacgc ctgccctccccagagcactg g 341 28 8 PRT Homo sapiens 28 Ser Ala Arg Asn Arg Gln LysArg 1 5 29 49 DNA Artificial Sequence Description of ArtificialSequencePCR primer 29 cgtacgaatt ccagaagcgc gtatggagtg ggtgtgggcgctcgtgctg 49 30 42 DNA Artificial Sequence Description of ArtificialSequencePCR primer 30 gcatgactag tcaaactgtt tcttgagggt ctgctttgac ag 4231 70 DNA Artificial Sequence Description of Artificial SequencePCRprimer 31 gcaacactag tgctagaaac cgtcagaaga gattcccaac cattcccttaagcaggcctt 60 ttgacaacgc 70 32 45 DNA Artificial Sequence Description ofArtificial SequencePCR primer 32 gctcaggatc ccgggctaga agccacagctgccctccaca gagcg 45 33 32 DNA Artificial Sequence Description ofArtificial SequencePCR primer 33 tcagctctag agagcgcgac tgcagggtga gc 3234 33 DNA Artificial Sequence Description of Artificial SequencePCRprimer 34 gaagcactag tcaaactgtt tcttgagggt ctg 33 35 1275 DNA Homosapiens 35 gaattccaga agcgcgtatg gagtgggtgt gggcgctcgt gctgctggcggctctgggag 60 gcggcagcgc cgagcgcgac tgcagggtga gcagcttcag agtcaaggagaacttcgaca 120 aggctcgttt ctctgggctc tggtatgcca tcgccaaaaa ggatcccgagggtctctttt 180 tgcaagacaa catcatcgct gagttttctg tcgacgagaa gggtcatatgagcgctacag 240 ccaagggacg agtccgtctt ctgagcaact gggaagtgtg tgcagacatggtgggcactt 300 tcacagatac agaagatcct gccaagttca agatgaagta ctggggtgtagcctcctttc 360 tccagcgagg aaacgatgac cactggatca tcgatacgga ctacgacaccttcgctctgc 420 agtattcctg ccgcctgcag aatctggatg gcacctgtgc agacagctactcctttgtgt 480 tttctcgtga ccccaatggc ctgaccccgg agacacggag gctggtgaggcagcgacagg 540 aggagctgtg cctagagagg cagtacagat ggatcgagca caatggttactgtcaaagca 600 gaccctcaag aaacagtttg actagtgcta gaaaccgtca gaagagattcccaaccattc 660 ccttaagcag gccttttgac aacgctatgc tccgcgccca tcgtctgcaccagctggcct 720 ttgacaccta ccaggagttt gaagaagcct atatcccaaa ggaacagaagtattcattcc 780 tgcagaaccc ccagacctcc ctctgtttct cagagtctat tccgacaccctccaacaggg 840 aggaaacaca acagaaatcc aacctagagc tgctccgcat ctccctgctgctcatccagt 900 cgtggctgga gcccgtgcag ttcctcagga gtgtcttcgc caacagcctggtgtacggcg 960 cctctgacag caacgtctat gacctcctaa aggacctaga ggaaggcatccaaacgctga 1020 tggggaggct ggaagatggc agcccccgga ctgggcagat cttcaagcagacctacagca 1080 agttcgacac aaactcacac aacgatgacg cactactcaa gaactacgggctgctctact 1140 gcttcaggaa ggacatggac aaggtcgaga cattcctgcg catcgtgcagtgccgctctg 1200 tggagggcag ctgtggcttc tagcccggga tcctgagaac ttcagggtgagtttggggac 1260 ccttgattgt tcttt 1275 36 401 PRT Homo sapiens 36 Met GluTrp Val Trp Ala Leu Val Leu Leu Ala Ala Leu Gly Gly Gly 1 5 10 15 SerAla Glu Arg Asp Cys Arg Val Ser Ser Phe Arg Val Lys Glu Asn 20 25 30 PheAsp Lys Ala Arg Phe Ser Gly Leu Trp Tyr Ala Ile Ala Lys Lys 35 40 45 AspPro Glu Gly Leu Phe Leu Gln Asp Asn Ile Ile Ala Glu Phe Ser 50 55 60 ValAsp Glu Lys Gly His Met Ser Ala Thr Ala Lys Gly Arg Val Arg 65 70 75 80Leu Leu Ser Asn Trp Glu Val Cys Ala Asp Met Val Gly Thr Phe Thr 85 90 95Asp Thr Glu Asp Pro Ala Lys Phe Lys Met Lys Tyr Trp Gly Val Ala 100 105110 Ser Phe Leu Gln Arg Gly Asn Asp Asp His Trp Ile Ile Asp Thr Asp 115120 125 Tyr Asp Thr Phe Ala Leu Gln Tyr Ser Cys Arg Leu Gln Asn Leu Asp130 135 140 Gly Thr Cys Ala Asp Ser Tyr Ser Phe Val Phe Ser Arg Asp ProAsn 145 150 155 160 Gly Leu Thr Pro Glu Thr Arg Arg Leu Val Arg Gln ArgGln Glu Glu 165 170 175 Leu Cys Leu Glu Arg Gln Tyr Arg Trp Ile Glu HisAsn Gly Tyr Cys 180 185 190 Gln Ser Arg Pro Ser Arg Asn Ser Leu Thr SerAla Arg Asn Arg Gln 195 200 205 Lys Arg Phe Pro Thr Ile Pro Leu Ser ArgPro Phe Asp Asn Ala Met 210 215 220 Leu Arg Ala His Arg Leu His Gln LeuAla Phe Asp Thr Tyr Gln Glu 225 230 235 240 Phe Glu Glu Ala Tyr Ile ProLys Glu Gln Lys Tyr Ser Phe Leu Gln 245 250 255 Asn Pro Gln Thr Ser LeuCys Phe Ser Glu Ser Ile Pro Thr Pro Ser 260 265 270 Asn Arg Glu Glu ThrGln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile 275 280 285 Ser Leu Leu LeuIle Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg 290 295 300 Ser Val PheAla Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val 305 310 315 320 TyrAsp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly 325 330 335Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr 340 345350 Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys 355360 365 Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu370 375 380 Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser CysGly 385 390 395 400 Phe 37 400 PRT Homo sapiens 37 Met Glu Trp Val TrpAla Leu Val Leu Leu Ala Ala Leu Gly Gly Gly 1 5 10 15 Ser Ala Glu ArgAsp Cys Arg Val Ser Ser Phe Arg Val Lys Glu Asn 20 25 30 Phe Asp Lys AlaArg Phe Ser Gly Leu Trp Tyr Ala Ile Ala Lys Lys 35 40 45 Asp Pro Glu GlyLeu Phe Leu Gln Asp Asn Ile Ile Ala Glu Phe Ser 50 55 60 Val Asp Glu LysGly His Met Ser Ala Thr Ala Lys Gly Arg Val Arg 65 70 75 80 Leu Leu SerAsn Trp Glu Val Cys Ala Asp Met Val Gly Thr Phe Thr 85 90 95 Asp Thr GluAsp Pro Ala Lys Phe Lys Met Lys Tyr Trp Gly Val Ala 100 105 110 Ser PheLeu Gln Arg Gly Asn Asp Asp His Trp Ile Ile Asp Thr Asp 115 120 125 TyrAsp Thr Phe Ala Leu Gln Tyr Ser Cys Arg Leu Gln Asn Leu Asp 130 135 140Gly Thr Cys Ala Asp Ser Tyr Ser Phe Val Phe Ser Arg Asp Pro Asn 145 150155 160 Gly Leu Thr Pro Glu Thr Arg Arg Leu Val Arg Gln Arg Gln Glu Glu165 170 175 Leu Cys Leu Glu Arg Gln Tyr Arg Trp Ile Glu His Asn Gly TyrCys 180 185 190 Gln Ser Arg Pro Ser Arg Asn Ser Leu Thr Ser Ala Arg AsnArg Gln 195 200 205 Lys Arg Phe Pro Thr Ile Pro Leu Ser Arg Pro Phe AspAsn Ala Met 210 215 220 Leu Arg Ala His Arg Leu His Gln Leu Ala Phe AspThr Tyr Gln Glu 225 230 235 240 Phe Glu Glu Ala Tyr Ile Pro Lys Glu GlnLys Tyr Ser Phe Leu Gln 245 250 255 Asn Pro Gln Thr Ser Leu Cys Phe SerGlu Ser Ile Pro Thr Pro Ser 260 265 270 Asn Arg Glu Glu Thr Gln Gln LysSer Asn Leu Glu Leu Leu Arg Ile 275 280 285 Ser Leu Leu Leu Ile Gln SerTrp Leu Glu Pro Val Gln Phe Leu Arg 290 295 300 Ser Val Phe Ala Asn SerLeu Val Tyr Gly Ala Ser Asp Ser Asn Val 305 310 315 320 Tyr Asp Leu LeuLys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly 325 330 335 Arg Leu GluAsp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr 340 345 350 Tyr SerLys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys 355 360 365 AsnTyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu 370 375 380Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly 385 390395 400 38 1204 DNA Homo sapiens 38 atggagtggg tgtgggcgct cgtgctgctggcggctctgg gaggcggcag cgccgagcgc 60 gactgcaggg tgagcagctt cagagtcaaggagaacttcg acaaggctcg tttctctggg 120 ctctggtatg ccatcgccaa aaaggatcccgagggtctct ttttgcaaga caacatcatc 180 gctgagtttt ctgtcgacga gaagggtcatatgagcgcta cagccaaggg acgagtccgt 240 cttctgagca actgggaagt gtgtgcagacatggtgggca ctttcacaga tacagaagat 300 cctgccaagt tcaagatgaa gtactggggtgtagcctcct ttctccagcg aggaaacgat 360 gaccactgga tcatcgatac ggactacgacaccttcgctc tgcagtattc ctgccgcctg 420 cagaatctgg atggcacctg tgcagacagctactcctttg tgttttctcg tgaccccaat 480 ggcctgaccc cggagacacg gaggctggtgaggcagcgac aggaggagct gtgcctagag 540 aggcagtaca gatggatcga gcacaatggttactgtcaaa gcagaccctc aagaaacagt 600 ttgactagtg ctagaaaccg tcagaagagattcccaacca ttcccttaag caggcctttt 660 gacaacgcta tgctccgcgc ccatcgtctgcaccagctgg cctttgacac ctaccaggag 720 tttgaagaag cctatatccc aaaggaacagaagtattcat tcctgcagaa cccccagacc 780 tccctctgtt tctcagagtc tattccgacaccctccaaca gggaggaaac acaacagaaa 840 tccaacctag agctgctccg catctccctgctgctcatcc agtcgtggct ggagcccgtg 900 cagttcctca ggagtgtctt cgccaacagcctggtgtacg gcgcctctga cagcaacgtc 960 tatgacctcc taaaggacct agaggaaggcatccaaacgc tgatggggag gctggaagat 1020 ggcagccccc ggactgggca gatcttcaagcagacctaca gcaagttcga cacaaactca 1080 cacaacgatg acgcactact caagaactacgggctgctct actgcttcag gaaggacatg 1140 gacaaggtcg agacattcct gcgcatcgtgcagtgccgct ctgtggaggc agctgtggtt 1200 ctag 1204

What is claimed:
 1. A cell containing a recombinant nucleic acidencoding a fusion protein comprising at least one conditional retentiondomain (“CRD”) and at least one additional domain that is heterologousthereto.
 2. A cell of claim 1 wherein the fusion protein contains morethan one CRD.
 3. A cell of claim 1 wherein the fusion protein moleculesform aggregates with one another in the absence of a ligand which bindsto the CRD.
 4. A cell of claim 1 wherein the CRD is derived from retinolbinding protein, FKBP, IgM or alpha1-antitrypsin.
 5. A cell of claim 4wherein the CRD comprises an FKBP domain with an amino acid replacementat F36 or W59.
 6. A cell of claim 5 wherein the CRD comprises an FKBPdomain containing the mutation F36M or W59V.
 7. A cell of claim 1wherein the heterologous domain of the fusion protein comprises thepolypeptide sequence of a protein of interest.
 8. A cell of claim 7wherein the protein of interest is a hormone, an endorphin, an antibodyor an immunogen.
 9. A cell of claim 8 wherein the protein of interest isselected from the group consisting of insulin, parathyroid hormone andbeta-endorphin.
 10. A cell of claim 1 wherein the fusion proteincomprises an enzymatic cleavage site.
 11. A cell of claim 10 wherein thecleavage site is a furin cleavage site.
 12. A cell of claim 11 whereinthe furin cleavage site comprises the amino acid sequence SARNRQKR (SEQID NO. 1).
 13. A cell of claim 1 wherein the fusion protein furthercomprises a secretory signal sequence.
 14. A cell of claim 13 whereinthe secretory signal sequence is the signal sequence from the humangrowth hormone gene.
 15. A cell of claim 1 wherein the fusion proteincomprises a secretory signal sequence, at least one conditionalretention domain, a furin cleavage site, and a polypeptide sequence ofinterest.
 16. A cell of claim 15 wherein the fusion protein comprises asecretory signal sequence from human growth hormone, three F36M FKBPdomains, a human stromelysin-3 furin cleavage site, and a polypeptidesequence of interest.
 17. A cell of claim 1 wherein the fusion proteincomprises a lysosomal targeting signal.
 18. A cell of claim 1 whereinthe cells are mammalian cells.
 19. A cell of claim 1 wherein the cellsare of human origin.
 20. A cell of claim 1 wherein the cells are primarycells.
 21. A cell of claim 1 wherein the cells are in an animal.